Human CD163 antibodies and uses thereof

ABSTRACT

Provided herein are antibodies and methods of use thereof. The antibodies as disclosed herein bind to CD163+ on cells, such as on macrophages. These antibodies can be used in methods of treatment, such as methods of treating cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/935,814, filed Jul. 22, 2020, now issued and U.S. Pat. No. 11,034,770on Jun. 15, 2021, which is a continuation of International ApplicationNo. PCT/US2020/042668, filed on Jul. 17, 2020, which claims priority toand benefit from U.S. Provisional Application Nos. 62/876,580, filedJul. 19, 2019, 62/876,579, filed Jul. 19, 2019, and 62/878,265, filedJul. 24, 2019, the entire contents of each are herein incorporated byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 21, 2021, isnamed ONR_003C1_SL.txt and is 70,484 bytes in size.

SUMMARY OF THE DISCLOSURE

Provided herein are antibodies, including antigen-binding fragments andother antigen-binding polypeptides, that are useful in the treatment ofcancer and other disorders. In some embodiments the antibodiesspecifically bind to M2 and M2-like immune-suppressive macrophages butnot to M1 and M1-like anti-tumor macrophages. The disclosed antibodiesbind to M2 and M2-like tumor-associated macrophages and modulate thephysical and functional characteristics of tumor-associated M2 andM2-like macrophages to relieve immunosuppression of the tumormicroenvironment and increase cytotoxic T-cell activation andproliferation, which promotes tumor cell killing. The antibody moleculesof the present disclosure specifically bind to human CD163 expressed onthe surface of M2 and M2-like macrophages.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody comprising a heavy chain variable region (VH)having at least 80% identity to amino acid sequence SEQ ID NO: 8.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody comprising a heavy chain variable region (VH)having at least 90% identity to amino acid sequence SEQ ID NO: 8.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody comprising a heavy chain variable region (VH)having at least 95% identity to amino acid sequence SEQ ID NO: 8.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody comprising a heavy chain variable region (VH)having at least 99% identity to amino acid sequence SEQ ID NO: 8.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody comprising a heavy chain variable region (VH)having at least 100% identity to amino acid sequence SEQ ID NO: 8. Insome embodiments, the antibody or the recombinant antibody furthercomprises a light chain variable region (VL) having at least 80%identity to amino acid sequence SEQ ID NO: 7. In some embodiments, theantibody or the recombinant antibody further comprises a light chainvariable region (VL) having at least 90% identity to amino acid sequenceSEQ ID NO: 7. In some embodiments, the antibody or the recombinantantibody further comprises a light chain variable region (VL) having atleast 95% identity to amino acid sequence SEQ ID NO: 7. In someembodiments, the antibody or the recombinant antibody further comprisesa light chain variable region (VL) having at least 99% identity to aminoacid sequence SEQ ID NO: 7. In some embodiments, the antibody or therecombinant antibody further comprises a light chain variable region(VL) having at least 100% identity to amino acid sequence SEQ ID NO: 7.

Disclosed herein, in certain embodiments, are a light chain variableregion (VL) having at least 80% identity to amino acid sequence SEQ IDNO: 7.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody comprising a light chain variable region (VL)having at least 90% identity to amino acid sequence SEQ ID NO: 7.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody comprising a light chain variable region (VL)having at least 95% identity to amino acid sequence SEQ ID NO: 7

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody comprising a light chain variable region (VL)having at least 99% identity to amino acid sequence SEQ ID NO: 7.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody comprising a light chain variable region (VL)having at least 100% identity to amino acid sequence SEQ ID NO: 7. Insome embodiments, the antibody or the recombinant antibody furthercomprises a heavy chain variable region (VH) having at least 80%identity to amino acid sequence SEQ ID NO: 8. In some embodiments, theantibody or the recombinant antibody further comprises a heavy chainvariable region (VH) having at least 90% identity to amino acid sequenceSEQ ID NO: 8. In some embodiments, the antibody or the recombinantantibody further comprises a heavy chain variable region (VH) having atleast 95% identity to amino acid sequence SEQ ID NO: 8. In someembodiments, the antibody or the recombinant antibody further comprisesa heavy chain variable region (VH) having at least 99% identity to aminoacid sequence SEQ ID NO: 8. In some embodiments, the antibody or therecombinant antibody further comprises a heavy chain variable region(VH) having at least 100% identity to amino acid sequence SEQ ID NO: 8.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody comprising a heavy chain variable region (V_(H))having at least 80% identity to amino acid sequence SEQ ID NO: 8 and alight chain variable region (V_(L)) having at least 80% identity toamino acid sequence SEQ ID NO: 7. In some embodiments, the antibody orthe recombinant antibody comprises the light chain variable region(V_(L)) having at least 85% identity to amino acid sequence SEQ ID NO:7. In some embodiments, the antibody or the recombinant antibodycomprises the light chain variable region (V_(L)) having at least 90%identity to amino acid sequence SEQ ID NO: 7. In some embodiments, theantibody or the recombinant antibody comprises the light chain variableregion (V_(L)) having at least 95% identity to amino acid sequence SEQID NO: 7. In some embodiments, the antibody or the recombinant antibodycomprises the light chain variable region (V_(L)) having at least 99%identity to amino acid sequence SEQ ID NO: 7. In some embodiments, theantibody or the recombinant antibody comprises the light chain variableregion (V_(L)) having at least 100% identity to amino acid sequence SEQID NO: 7. In some embodiments, the antibody or the recombinant antibodycomprises the heavy chain variable region (V_(H)) having at least 85%identity to amino acid sequence SEQ ID NO: 8. In some embodiments, theantibody or the recombinant antibody comprises the heavy chain variableregion (V_(H)) having at least 90% identity to amino acid sequence SEQID NO: 8. In some embodiments, the antibody or the recombinant antibodycomprises the heavy chain variable region (V_(H)) having at least 95%identity to amino acid sequence SEQ ID NO: 8. In some embodiments, theantibody or the recombinant antibody comprises the heavy chain variableregion (V_(H)) having at least 99% identity to amino acid sequence SEQID NO: 8. In some embodiments, the antibody or the recombinant antibodycomprises the heavy chain variable region (V_(H)) having at least 100%identity to amino acid sequence SEQ ID NO: 8.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody comprising a heavy chain sequence comprising atleast one of complementarity determining region (CDR) H1 having at least80% identity to amino acid sequence SEQ ID NO: 4, a CDR H2 having atleast 80% identity to amino acid sequence SEQ ID NO: 5, and a CDR H3having at least 80% identity to amino acid sequence SEQ ID NO: 6, and alight chain sequence comprising a CDR L1 having at least 80% identity toamino acid sequence SEQ ID NO: 1, a CDR L2 having at least 80% identityto amino acid sequence SEQ ID NO: 2, and a CDR L3 having at least 80%identity to amino acid sequence SEQ ID NO: 3. In some embodiments, theantibody or the recombinant antibody comprises the light chain sequencecomprising the CDR L1 having at least 85% identity to amino acidsequence SEQ ID NO: 1, the CDR L2 having at least 85% identity to aminoacid sequence SEQ ID NO: 2, and the CDR L3 having at least 85% identityto amino acid sequence SEQ ID NO: 3. In some embodiments, the antibodyor the recombinant antibody comprises the light chain sequencecomprising the CDR L1 having at least 90% identity to amino acidsequence SEQ ID NO: 1, the CDR L2 having at least 90% identity to aminoacid sequence SEQ ID NO: 2, and the CDR L3 having at least 90% identityto amino acid sequence SEQ ID NO: 3. In some embodiments, the antibodyor the recombinant antibody comprises light chain sequence comprisingthe CDR L1 having at least 95% identity to amino acid sequence SEQ IDNO: 1, the CDR L2 having at least 95% identity to amino acid sequenceSEQ ID NO: 2, and the CDR L3 having at least 95% identity to amino acidsequence SEQ ID NO: 3. In some embodiments, the antibody or therecombinant antibody comprises the light chain sequence comprising theCDR L1 having at least 99% identity to amino acid sequence SEQ ID NO: 1,the CDR L2 having at least 99% identity to amino acid sequence SEQ IDNO: 2, and the CDR L3 having at least 99% identity to amino acidsequence SEQ ID NO: 3. In some embodiments, the antibody or therecombinant antibody comprises the light chain sequence comprising theCDR L1 having at least 100% identity to amino acid sequence SEQ ID NO:1, the CDR L2 having at least 100% identity to amino acid sequence SEQID NO: 2, and the CDR L3 having at least 100% identity to amino acidsequence SEQ ID NO: 3. In some embodiments, the antibody or therecombinant antibody comprises the heavy chain sequence comprising a theCDR H1 having at least 85% identity to amino acid sequence SEQ ID NO: 4,the CDR H2 having at least 85% identity to amino acid sequence SEQ IDNO: 5, and the CDR H3 having at least 85% identity to amino acidsequence SEQ ID NO: 6. In some embodiments, the antibody or therecombinant antibody comprises the heavy chain sequence comprising a theCDR H1 having at least 90% identity to amino acid sequence SEQ ID NO: 4,the CDR H2 having at least 90% identity to amino acid sequence SEQ IDNO: 5, and the CDR H3 having at least 90% identity to amino acidsequence SEQ ID NO: 6. In some embodiments, the antibody or therecombinant antibody comprises the heavy chain sequence comprising a theCDR H1 having at least 95% identity to amino acid sequence SEQ ID NO: 4,the CDR H2 having at least 95% identity to amino acid sequence SEQ IDNO: 5, and the CDR H3 having at least 95% identity to amino acidsequence SEQ ID NO: 6. In some embodiments, the antibody or therecombinant antibody comprises the heavy chain sequence comprising a theCDR H1 having at least 99% identity to amino acid sequence SEQ ID NO: 4,the CDR H2 having at least 99% identity to amino acid sequence SEQ IDNO: 5, and the CDR H3 having at least 99% identity to amino acidsequence SEQ ID NO: 6. In some embodiments, the antibody or therecombinant antibody comprises the heavy chain sequence comprising a theCDR H1 having at least 100% identity to amino acid sequence SEQ ID NO:4, the CDR H2 having at least 100% identity to amino acid sequence SEQID NO: 5, and the CDR H3 having at least 100% identity to amino acidsequence SEQ ID NO: 6.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody, comprising a heavy chain sequence comprising atleast one of complementarity determining region (CDR) H1 having at least80% identity to amino acid sequence SEQ ID NO: 4, a CDR H2 having atleast 80% identity to amino acid sequence SEQ ID NO: 5, and a CDR H3having at least 80% identity to amino acid sequence SEQ ID NO: 6.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody, comprising a heavy chain sequence comprising atleast one of complementarity determining region (CDR) H1 having at least90% identity to amino acid sequence SEQ ID NO: 4, a CDR H2 having atleast 90% identity to amino acid sequence SEQ ID NO: 5, and a CDR H3having at least 90% identity to amino acid sequence SEQ ID NO: 6.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody, comprising a heavy chain sequence comprising atleast one of complementarity determining region (CDR) H1 having at least95% identity to amino acid sequence SEQ ID NO: 4, a CDR H2 having atleast 95% identity to amino acid sequence SEQ ID NO: 5, and a CDR H3having at least 95% identity to amino acid sequence SEQ ID NO: 6.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody, comprising a heavy chain sequence comprising atleast one of complementarity determining region (CDR) H1 having at least99% identity to amino acid sequence SEQ ID NO: 4, a CDR H2 having atleast 99% identity to amino acid sequence SEQ ID NO: 5, and a CDR H3having at least 99% identity to amino acid sequence SEQ ID NO: 6.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody, comprising a heavy chain sequence comprising atleast one of complementarity determining region (CDR) H1 having at least100% identity to amino acid sequence SEQ ID NO: 4, a CDR H2 having atleast 100% identity to amino acid sequence SEQ ID NO: 5, and a CDR H3having at least 100% identity to amino acid sequence SEQ ID NO: 6.

In some embodiments, the antibody or the recombinant antibody furthercomprises a light chain sequence comprising at least one complementaritydetermining region (CDR) L1 having at least 80% identity to amino acidsequence SEQ ID NO: 1, a CDR L2 having at least 80% identity to aminoacid sequence SEQ ID NO: 2, and a CDR L3 having at least 80% identity toamino acid sequence SEQ ID NO: 3. In some embodiments, the CDR L1 has atleast 90% identity to amino acid sequence SEQ ID NO: 1, the CDR L2 hasat least 90% identity to amino acid sequence SEQ ID NO: 2, and the CDRL3 has at least 90% identity to amino acid sequence SEQ ID NO: 3. Insome embodiments, the CDR L1 has at least 95% identity to amino acidsequence SEQ ID NO: 1, the CDR L2 has at least 95% identity to aminoacid sequence SEQ ID NO: 2, and the CDR L3 has at least 95% identity toamino acid sequence SEQ ID NO: 3. In some embodiments, the CDR L1 has atleast 99% identity to amino acid sequence SEQ ID NO: 1, the CDR L2 hasat least 99% identity to amino acid sequence SEQ ID NO: 2, and the CDRL3 has at least 99% identity to amino acid sequence SEQ ID NO: 3. Insome embodiments, the CDR L1 has at least 100% identity to amino acidsequence SEQ ID NO: 1, the CDR L2 has at least 100% identity to aminoacid sequence SEQ ID NO: 2, and the CDR L3 has at least 100% identity toamino acid sequence SEQ ID NO: 3. In some embodiments, the light chainvariable region (VL) has at least 80% identity to amino acid sequenceSEQ ID NO: 7.

In some embodiments, the antibody or the recombinant antibody furthercomprises a light chain variable region (VL) having at least 80%identity to amino acid sequence SEQ ID NO: 7. In some embodiments, theantibody or the recombinant antibody comprises a heavy chain variableregion (VH) having at least 80% identity to amino acid sequence SEQ IDNO: 8.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody, comprising a light chain sequence comprising acomplementarity determining region (CDR) L1 having at least 80% identityto amino acid sequence SEQ ID NO: 1, a CDR L2 having at least 80%identity to amino acid sequence SEQ ID NO: 2, and a CDR L3 having atleast 80% identity to amino acid sequence SEQ ID NO: 3.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody, comprising a light chain sequence comprising acomplementarity determining region (CDR) L1 having at least 90% identityto amino acid sequence SEQ ID NO: 1, a CDR L2 having at least 90%identity to amino acid sequence SEQ ID NO: 2, and a CDR L3 having atleast 90% identity to amino acid sequence SEQ ID NO: 3.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody, comprising a light chain sequence comprising acomplementarity determining region (CDR) L1 having at least 95% identityto amino acid sequence SEQ ID NO: 1, a CDR L2 having at least 95%identity to amino acid sequence SEQ ID NO: 2, and a CDR L3 having atleast 95% identity to amino acid sequence SEQ ID NO: 3.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody, comprising a light chain sequence comprising acomplementarity determining region (CDR) L1 having at least 99% identityto amino acid sequence SEQ ID NO: 1, a CDR L2 having at least 99%identity to amino acid sequence SEQ ID NO: 2, and a CDR L3 having atleast 99% identity to amino acid sequence SEQ ID NO: 3.

Disclosed herein, in certain embodiments, are an antibody or arecombinant antibody, comprising a light chain sequence comprising acomplementarity determining region (CDR) L1 having at least 100%identity to amino acid sequence SEQ ID NO: 1, a CDR L2 having at least100% identity to amino acid sequence SEQ ID NO: 2, and a CDR L3 havingat least 100% identity to amino acid sequence SEQ ID NO: 3.

In some embodiments, the antibody or the recombinant antibody furthercomprises a heavy chain sequence comprising at least one ofcomplementarity determining region (CDR) H1 having at least 80% identityto amino acid sequence SEQ ID NO: 4, a CDR H2 having at least 80%identity to amino acid sequence SEQ ID NO: 5, and a CDR H3 having atleast 80% identity to amino acid sequence SEQ ID NO: 6. In someembodiments, the CDR H1 has at least 90% identity to amino acid sequenceSEQ ID NO: 4, the CDR H2 has at least 90% identity to amino acidsequence SEQ ID NO: 5, and the CDR H3 has at least 90% identity to aminoacid sequence SEQ ID NO: 6. In some embodiments, the CDR H1 has at least95% identity to amino acid sequence SEQ ID NO: 4, the CDR H2 has atleast 95% identity to amino acid sequence SEQ ID NO: 5, and the CDR H3has at least 95% identity to amino acid sequence SEQ ID NO: 6. In someembodiments, the CDR H1 has at least 99% identity to amino acid sequenceSEQ ID NO: 4, the CDR H2 has at least 99% identity to amino acidsequence SEQ ID NO: 5, and the CDR H3 has at least 99% identity to aminoacid sequence SEQ ID NO: 6. In some embodiments, the CDR H1 has at least100% identity to amino acid sequence SEQ ID NO: 4, the CDR H2 has atleast 100% identity to amino acid sequence SEQ ID NO: 5, and the CDR H3has at least 100% identity to amino acid sequence SEQ ID NO: 6. In someembodiments, the heavy chain variable region (VH) has at least 80%identity to amino acid sequence SEQ ID NO: 8.

In some embodiments, the antibody or the recombinant antibody furthercomprises a heavy chain variable region (VH) having at least 80%identity to amino acid sequence SEQ ID NO: 8. In some embodiments, thelight chain variable region (VL) having at least 80% identity to aminoacid sequence SEQ ID NO: 7.

In some embodiments, the antibody or the recombinant antibody furthercomprises a human heavy chain constant region or a human light chainconstant region. In some embodiments, the human heavy chain constantregion is IgG1 or IgG4 or a fragment thereof. In some embodiments, theheavy chain has at least 80% identity to the amino acid sequence of SEQID NO: 10. In some embodiments, the light chain has at least 80%identity to the amino acid sequence of SEQ ID NO: 9. In someembodiments, wherein the heavy chain has at least 80% identity to theamino acid sequence of SEQ ID NO: 12. In some embodiments, the heavychain has at least 80% identity to the amino acid sequence of SEQ ID NO:11. In some embodiments, heavy chain has at least 80% identity to theamino acid sequence of SEQ ID NO: 13. In some embodiments, the antibodyor the recombinant antibody comprises a human variable framework regionand a murine constant region. In some embodiments, the antibody or therecombinant antibody further comprises a murine heavy chain constantregion or a murine light chain constant region. In some embodiments, theantibody or the murine heavy chain constant region is IgG2A. In someembodiments, the heavy chain has at least 80% identity to the amino acidsequence of SEQ ID NO: 15. In some embodiments, the heavy chain has atleast 80% identity to the amino acid sequence of SEQ ID NO: 16. In someembodiments, the light chain has at least 80% identity to the amino acidsequence of SEQ ID NO: 14. In some embodiments, the antibody or therecombinant antibody is an antibody fragment comprising a single heavychain, a single light chain, Fab, Fab′, F(ab)′, F(ab′)2, Fd, scFv, avariable heavy domain, a variable light domain, a variable NAR domain,bi-specific scFv, a bi-specific Fab2, a tri-specific Fab3, a singlechain binding polypeptide, a dAb fragment, or a diabody.

In some embodiments, the antibody or the recombinant antibodyspecifically binds to a CD163 protein expressed on an immunosuppressivehuman myeloid cell, wherein binding of the antibody or the recombinantantibody to the myeloid cell promotes an immune cell function asmeasured by one or both of the following parameters: (i) activation of aCD4⁺ T cell, CD8⁺ T cell, NK cell, or any combination thereof; and (ii)proliferation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any combinationthereof. In some embodiments, wherein activation of a CD4⁺ T cell, CD8⁺T cell, NK cell, or any combination thereof is measured as an increasedlevel of IFN-γ, TNF-α, or perforin, or any combination thereof. In someembodiments, the immunosuppressive human myeloid cell is a macrophage ora myeloid-derived suppressor cell. In some embodiments, the immune cellfunction is in a tumor microenvironment. In some embodiments, the immunecell function is in vivo. In some embodiments, the antibody or therecombinant antibody specifically binds to a CD163 protein expressed ona human macrophage, wherein binding of the antibody to the macrophageincreases an immunostimulatory activity in a tumor microenvironment. Insome embodiments, the tumor microenvironment is in vivo. In someembodiments, binding of the antibody or the recombinant antibody to themacrophage reduces an immunosuppression activity of the macrophage. Insome embodiments, binding of the antibody or the recombinant antibody tothe macrophage reduces a tumor promoting activity of the macrophage. Insome embodiments, the macrophage is a tumor-associated macrophage. Insome embodiments, the antibody or recombinant antibody alters expressionof at least one marker on the macrophage. In some embodiments, the CD163protein is a glycoform of CD163. In some embodiments, the CD163 proteinis a 150 kDa glycoform of CD163. In some embodiments, the antibody doesnot specifically bind to a 130 kDa glycoform of CD163 expressed by thehuman macrophage.

In some embodiments, the antibody or the recombinant antibody has aconstant domain that enables binding to an Fc receptor. In someembodiments, the Fc receptor is expressed on the macrophage. In someembodiments, the antibody or the recombinant antibody has an antibodyfragment comprising a single heavy chain, a single light chain, Fab,Fab′, F(ab)′, F(ab′)2, Fd, scFv, a variable heavy domain, a variablelight domain, a variable NAR domain, bi-specific scFv, a bi-specificFab2, a tri-specific Fab3, a single chain binding polypeptide, a dAbfragment, or a diabody. In some embodiments, at least one marker on thehuman macrophage is CD16, CD64, TLR2, or Siglec-15. In some embodiments,the human macrophage is an M2 macrophage or a M2-like macrophage. Insome embodiments, the human macrophage is an M2a, M2b, M2c, or M2dmacrophage. In some embodiments, the CD163 protein is a component of acell surface complex comprising at least one other protein expressed bythe macrophage. In some embodiments, the at least one other protein is agalectin-1 protein, a LILRB2 protein, a casein kinase II protein, or anycombination thereof. In some embodiments, upon binding with the CD163protein, the antibody or the recombinant antibody is internalized by thehuman macrophage. In some embodiments, binding to the CD163 protein isnot cytotoxic to the macrophage. In some embodiments, binding to theCD163 protein promotes CD4⁺ T cell activation, CD4⁺ T cellproliferation, or both CD4⁺ T cell activation and proliferation. In someembodiments, binding to the CD163 protein promotes expression of CD69,ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by CD4⁺ Tcells. In some embodiments, binding to the CD163 protein promotes CD8⁺ Tcell activation, CD8⁺ T cell proliferation, or both CD8⁺ T cellactivation and proliferation. In some embodiments, binding to the CD163protein promotes expression of ICOS, OX40, PD1, LAG3, CTLA4, or anycombination thereof by CD8⁺ T cells. In some embodiments, binding to theCD163 protein reduces immunosuppression in a tumor microenvironment. Insome embodiments, binding to the CD163 protein promotes tumor cellkilling in a tumor microenvironment. In some embodiments, binding to theCD163 protein promotes cytotoxic lymphocyte-mediated killing of cancercells. In some embodiments, binding to the CD163 protein promotes NKcell-mediated tumor cell killing. In some embodiments, binding to theCD163 protein promotes expression of IL-2 by T cells. In someembodiments, binding to the CD163 protein increases CD4⁺ T cells, CD196⁻T cells, CXCR3⁺ T cells, CCR4⁻ T cells, or any combination thereof.

In some embodiments, binding to CD163 reduces immunosuppression in atumor microenvironment caused by a macrophage. In some embodiments, theantibody or the recombinant antibody specifically binds to a CD163protein expressed on a human macrophage, wherein binding results in atleast one of the following effects: (a) reduced expression of at leastone marker by the macrophage, wherein the at least one marker is CD16,CD64, TLR2, or Siglec-15; (b) internalization of the antibody by themacrophage; (c) activation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, orany combination thereof; (d) proliferation of a CD4⁺ T cell, CD8⁺ Tcell, NK cell, or any combination thereof; and (e) promotion of tumorcell killing in a tumor microenvironment. In some embodiments, thebinding results in: two or more of (a) through (e); three or more of (a)through (e); four or more of (a) through (e); or all of (a) through (e).In some embodiments, the antibody or the recombinant antibodyspecifically binds to a CD163⁺ immunosuppressive myeloid cell in a tumormicroenvironment, wherein the binding reduces suppression of cytotoxic Tcell-mediated killing of tumor cells in the tumor microenvironment. Insome embodiments, the antibody or the recombinant antibody specificallybinds to a CD163 protein expressed on a human tumor-associatedmacrophage and reduces expression of CD16, CD64, TLR2, Siglec-15, or acombination thereof by the macrophage, for use in a method of treatingcancer. In some embodiments, binding of the antibody or the recombinantantibody to the macrophage modulates an immune function of a cell in atumor microenvironment. In some embodiments, binding of the antibody orthe recombinant antibody to the macrophage promotes an anti-tumor immunefunction.

In some embodiments, the antibody or the recombinant antibodyspecifically binds to a CD163 epitope comprising amino acid sequence ofSEQ ID NO: 18. In some embodiments, the antibody or the recombinantantibody specifically binds to a CD163 epitope comprising amino acidsequence of SEQ ID NO: 19. In some embodiments, the antibody or therecombinant antibody specifically binds to a CD163 epitope comprisingamino acid sequence of SEQ ID NO: 20. In some embodiments, the antibodyor the recombinant antibody specifically binds to a CD163 epitopecomprising each of amino acid sequence SEQ ID NO: 18, SEQ ID NO: 19, andSEQ ID NO: 20. In some embodiments, the antibody or the recombinantantibody specifically binds to CD163 with a K_(D) from 1 nM to 100 nM.In some embodiments, the antibody or the recombinant antibodyspecifically binds to CD163 with a K_(D) from 1 nM to 50 nM. In someembodiments, the antibody or the recombinant antibody specifically bindsto CD163 with a K_(D) from 1 nM to 10 nM. In some embodiments, the CD163is human CD163. In some embodiments, the antibody or the recombinantantibody specifically binds to M2c macrophages with a K_(D) from 1 nM to100 nM. In some embodiments, the antibody or the recombinant antibodyspecifically binds to M2c macrophages with a K_(D) from 1 nM to 50 nM.In some embodiments, the antibody or the recombinant antibodyspecifically binds to M2c macrophages with a K_(D) from 1 nM to 10 nM.In some embodiments, the M2c macrophages are human M2c macrophages.

Disclosed herein, in certain embodiments, are a composition comprisingan antibody or a recombinant antibody and an excipient.

Disclosed herein, in certain embodiments, are a pharmaceuticalcomposition, comprising an antibody or a recombinant antibody of any oneof the previous embodiments and a pharmaceutically acceptable carrier.

Disclosed herein, in certain embodiments, are a use of an antibody or arecombinant antibody of any one of the previous embodiments for themanufacture of a medicament for treating cancer in a human subject.

Disclosed herein, in certain embodiments, are a use of an antibody or arecombinant antibody of any one of the previous embodiments for themanufacture of a medicament that reduces immunosuppression by atumor-associated macrophage in a human subject having a cancer.

Disclosed herein, in certain embodiments, are a use of an antibody or arecombinant antibody of any one of the previous embodiments for themanufacture of a medicament that promotes T cell-mediated tumor cellkilling in a human subject having a cancer.

Disclosed herein, in certain embodiments, are a method of promoting animmune cell function, the method comprising: administering an antibodyor a recombinant antibody of any one of the previous embodiments to anindividual in need thereof, and promoting an immune cell function asmeasured by one or both of the following parameters: (i) activation of aCD4⁺ T cell, CD8⁺ T cell, NK cell, or any combination thereof; and (ii)proliferation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any combinationthereof. In some embodiments, the activation of a CD4⁺ T cell, CD8⁺ Tcell, NK cell, or any combination thereof is measured as an increasedlevel of IFN-γ, TNF-α, or perforin, or any combination thereof. In someembodiments, the immunosuppressive human myeloid cell is a macrophage.In some embodiments, the immunosuppressive human myeloid cell is amyeloid-derived suppressor cell. In some embodiments, the antibody orthe recombinant antibody is an antibody or a recombinant antibody of anyone of the previous embodiments.

Disclosed herein, in certain embodiments, are a method of treatingcancer in an individual in need thereof, the method comprisingadministering to the individual a therapeutically effective amount of anantibody or a recombinant antibody of any one of the previousembodiments, thereby treating the cancer in the individual.

Disclosed herein, in certain embodiments, are a method of treatingcancer in an individual in need thereof, the method comprisingadministering to the individual a therapeutically effective amount of anantibody or a recombinant antibody of any one of the previousembodiments, whereby immunosuppression by a tumor-associated macrophagein the individual is reduced.

Disclosed herein, in certain embodiments, are a method of treatingcancer in an individual in need thereof, the method comprisingadministering to the individual a therapeutically effective amount of anantibody or a recombinant antibody of any one of the previousembodiments, whereby T cell-mediated tumor cell killing in theindividual is increased.

Disclosed herein, in certain embodiments, are a method of reducing atumor-promoting activity of a tumor-associated macrophage in anindividual in need thereof, the method comprising administering to theindividual an amount of a pharmaceutical composition of any one of theprevious embodiments that is effective to modulate a CD4⁺ T cellactivation, CD4⁺ T cell proliferation, CD8⁺ T cell activation, CD8⁺ Tcell proliferation, or any combination thereof in the tumormicroenvironment.

Disclosed herein, in certain embodiments, are a method of promotinglymphocyte-mediated tumor cell killing in an individual in need thereof,the method comprising administering to the individual an effectiveamount of an antibody or a recombinant antibody of any one of theprevious embodiments or a pharmaceutical composition of any one of theprevious embodiments.

In some embodiments, the method further comprises promoting tumor cellkilling in the tumor microenvironment. In some embodiments, the canceris a lung cancer or sarcoma. In some embodiments, the lung cancer is alung carcinoma or lung adenocarcinoma. In some embodiments, the methodfurther comprises administering to the individual an additionalanticancer therapeutic or anticancer therapy. In some embodiments, theadditional anticancer therapy is surgical therapy, chemotherapy,radiation therapy, cryotherapy, hormonal therapy, immunotherapy, andcytokine therapy, and combinations thereof. In some embodiments, theadditional anticancer therapy is an immunotherapy. In some embodiments,the immunotherapy is a composition comprising a checkpoint inhibitor.

Disclosed herein, in certain embodiments, are a method of modulating anactivity of a tumor-associated macrophage in a tumor microenvironment,the method comprising contacting the tumor-associated macrophage with anantibody or a recombinant antibody that binds to a CD163-expressinghuman macrophage and comprises at least one of the following effects:(a) the binding of the antibody reduces expression of at least onemarker on the macrophage, wherein the at least one marker is CD16, CD64,TLR2, or Siglec-15; (b) upon binding of the antibody with the CD163protein, the antibody is internalized by the human macrophage; (c) thebinding of the antibody is not cytotoxic to the macrophage; (d) thebinding of the antibody or the recombinant antibody produces IFN-γ,TNF-α, perforin, or any combination thereof by a CD4⁺ T cell, CD8⁺ Tcell, NK cell, or any combination thereof; (e) the binding of theantibody or the recombinant antibody promotes an activation of a CD4⁺ Tcell, CD8⁺ T cell, NK cell, or any combination thereof; (f) the bindingof the antibody or the recombinant antibody promotes a proliferation ofa CD4⁺ T cell, CD8⁺ T cell, NK cell, or any combination thereof; and (g)the binding of the antibody or the recombinant antibody promotes tumorcell killing in the tumor microenvironment. In some embodiments, thebinding results in: two or more of (a) through (g); three or more of (a)through (g); four or more of (a) through (g); five or more of (a)through (g); six or more of (a) through (g); or all of (a) through (g).

In some embodiments, the method occurs in a tumor microenvironment. Insome embodiments, the method occurs in vivo. In some embodiments,binding of the antibody or the recombinant antibody to the macrophagemodulates an immune function of a cell in a tumor microenvironment. Insome embodiments, binding of the antibody or the recombinant antibody tothe macrophage promotes an anti-tumor immune function. In someembodiments, the antibody or the recombinant antibody comprises aconstant domain and the constant domain binds to an Fc receptor. In someembodiments, the Fc receptor is expressed on the macrophage. In someembodiments, the method further comprises internalizing the antibody orthe recombinant antibody by the human macrophage upon binding with theCD163 protein. In some embodiments, binding to the CD163 protein is notcytotoxic to the human macrophage. In some embodiments, the methodfurther comprises promoting expression of CD69, ICOS, OX40, PD1, LAG3,CTLA4, or any combination thereof by CD4⁺ T cells. In some embodiments,the method further comprises promoting expression of ICOS, OX40, PD1,LAG3, CTLA4, or any combination thereof by CD8⁺ T cells. In someembodiments, the method comprises reducing immunosuppression in a tumormicroenvironment. In some embodiments, the method comprises promotingcytotoxic lymphocyte-mediated killing of cancer cells. In someembodiments, the method comprises promoting NK cell-mediated tumor cellkilling. In some embodiments, the method comprises promoting expressionof IL-2 by T cells. In some embodiments, the method comprises increasingCD4⁺ T cells, CD196⁻ T cells, CXCR3⁺ T cells, CCR4⁻ T cells, or anycombination thereof.

Disclosed herein, in certain embodiments, is an antibody thatspecifically binds to a CD163 protein expressed on a immunosuppressivehuman myeloid cell, wherein binding of the antibody to the myeloid cellpromotes an immune cell function as measured by one or both of thefollowing parameters: (i) activation of a CD4⁺ T cell, CD8⁺ T cell, NKcell, or any combination thereof; and (ii) proliferation of a CD4⁺ Tcell, CD8⁺ T cell, NK cell, or any combination thereof. In someembodiments, the immunosuppressive human myeloid cell is a macrophage ora myeloid-derived suppressor cell. In some embodiments, the activationof a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any combination thereof ismeasured as increased production of IFN-γ, TNF-α, perforin, or anycombination thereof.

Disclosed herein, in certain embodiments, is an antibody thatspecifically binds to a CD163 protein expressed on a human macrophage,wherein binding of the antibody to the macrophage promotes an immunecell function as measured by one or both of the following parameters:(i) activation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or anycombination thereof and (ii) proliferation of a CD4⁺ T cell, CD8⁺ Tcell, NK cell, or any combination thereof. In some embodiments, theimmune cell function is in a tumor microenvironment. In someembodiments, the immune cell function is in vivo. In some embodiments,the activation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or anycombination thereof is measured as increased production of IFN-γ, TNF-α,perforin, or any combination thereof.

Disclosed herein, in certain embodiments, is an antibody thatspecifically binds to a CD163 protein expressed on a human macrophage,wherein binding of the antibody to the macrophage increases animmunostimulatory activity in a tumor microenvironment. In someembodiments, the tumor microenvironment is in vivo. In some embodiments,binding of the antibody to the macrophage reduces an immunosuppressionactivity of the macrophage. In some embodiments, binding of the antibodyto the macrophage reduces a tumor promoting activity of the macrophage.In some embodiments, the macrophage is a tumor-associated macrophage. Insome embodiments, the antibody alters expression of at least one markeron the macrophage. In some embodiments, the CD163 protein is a glycoformof CD163. In some embodiments, the CD163 protein is a 150 kDa glycoformof CD163. In some embodiments, the antibody does not specifically bindto a 130 kDa glycoform of CD163 expressed by the human macrophage. Insome embodiments, the antibody has a constant domain that enablesbinding to an Fc receptor. In some embodiments, the Fc receptor isexpressed on the macrophage. In some embodiments, an antibody fragmentcomprises a single heavy chain, a single light chain, Fab, Fab′, F(ab)′,F(ab′)₂, Fd, scFv, a variable heavy domain, a variable light domain, avariable NAR domain, bi-specific scFv, a bi-specific Fab₂, atri-specific Fab₃, a single chain binding polypeptide, a dAb fragment,or a diabody. In some embodiments, at least one marker on the humanmacrophage is CD16, CD64, TLR2, or Siglec-15. In some embodiments, thehuman macrophage is an M2 macrophage or a M2-like macrophage. In someembodiments, the human macrophage is an M2a, M2b, M2c, or M2dmacrophage. In some embodiments, the CD163 protein is a component of acell surface complex comprising at least one other protein expressed bythe macrophage. In some embodiments, the at least one other protein is agalectin-1 protein, a LILRB2 protein, a casein kinase II protein, or anycombination thereof. In some embodiments, upon binding with the CD163protein, the antibody is internalized by the human macrophage. In someembodiments, binding to the CD163 protein is not cytotoxic to themacrophage. In some embodiments, binding to the CD163 protein promotesCD4⁺ T cell activation, CD4⁺ T cell proliferation, or both CD4⁺ T cellactivation and proliferation. In some embodiments, binding to the CD163protein promotes expression of CD69, ICOS, OX40, PD1, LAG3, CTLA4, orany combination thereof by CD4⁺ T cells. In some embodiments, binding tothe CD163 protein promotes CD8⁺ T cell activation, CD8⁺ T cellproliferation, or both CD8⁺ T cell activation and proliferation. In someembodiments, binding to the CD163 protein promotes expression of ICOS,OX40, PD1, LAG3, CTLA4, or any combination thereof by CD8⁺ T cells. Insome embodiments, binding to the CD163 protein reduces immunosuppressionin a tumor microenvironment. In some embodiments, binding to the CD163protein promotes tumor cell killing in a tumor microenvironment. In someembodiments, binding to the CD163 protein promotes cytotoxiclymphocyte-mediated killing of cancer cells. In some embodiments,binding to the CD163 protein promotes NK cell-mediated tumor cellkilling. In some embodiments, binding to the CD163 protein promotesexpression of IL-2 by T cells. In some embodiments, binding to the CD163protein increases CD4⁺ T cells, CD196⁻ T cells, CXCR3⁺ T cells, CCR4⁻ Tcells, or any combination thereof. In some embodiments, binding to CD163reduces immunosuppression in a tumor microenvironment caused by amacrophage.

Disclosed herein, in certain embodiments, is an antibody thatspecifically binds to a CD163 protein expressed on a human macrophage,wherein binding results in at least one of the following effects: (a)reduced expression of at least one marker by the macrophage, wherein theat least one marker is CD16, CD64, TLR2, or Siglec-15; (b)internalization of the antibody by the macrophage; (c) activation of aCD4⁺ T cell, CD8⁺ T cell, NK cell, or any combination thereof; (d)proliferation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any combinationthereof; and (e) promotion of tumor cell killing in a tumormicroenvironment. In some embodiments, the binding results in: two ormore of (a) through (e); three or more of (a) through (e); four or moreof (a) through (e); or all of (a) through (e). In some embodiments, theactivation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any combinationthereof is measured as increased production of IFN-γ, TNF-α, perforin,or any combination thereof.

Disclosed herein, in certain embodiments, is an antibody thatspecifically binds to a CD163⁺ immunosuppressive myeloid cell in a tumormicroenvironment, wherein the binding reduces suppression of cytotoxic Tcell-mediated killing of tumor cells in the tumor microenvironment.

Disclosed herein, in certain embodiments, is an antibody thatspecifically binds to a CD163 protein expressed on a humantumor-associated macrophage and reduces expression of CD16, CD64, TLR2,Siglec-15, or a combination thereof by the macrophage, for use in amethod of treating cancer. In some embodiments, binding of the antibodyto the macrophage modulates an immune function of a cell in a tumormicroenvironment. In some embodiments, binding of the antibody to themacrophage promotes an anti-tumor immune function.

In some embodiments, a composition comprises an antibody according toany embodiment as disclosed herein, and an excipient.

In some embodiments, a pharmaceutical composition comprises an antibodyaccording to any embodiment as disclosed herein and a pharmaceuticallyacceptable carrier.

In some embodiments, a use of an antibody as disclosed herein is for themanufacture of a medicament for treating cancer in a human subject.

In some embodiments, a use of an antibody as disclosed herein is for themanufacture of a medicament that reduces immunosuppression by atumor-associated macrophage in a human subject having a cancer.

In some embodiments, a use of an antibody as disclosed herein is for themanufacture of a medicament that promotes T cell-mediated tumor cellkilling in a human subject having a cancer.

Disclosed herein, in certain embodiments, is a method of promoting animmune cell function, the method comprising: specifically binding anantibody to a CD163 protein expressed on an immunosuppressive humanmyeloid cell; and promoting an immune cell function as measured by oneor both of the following parameters: (i) activation of a CD4⁺ T cell,CD8⁺ T cell, NK cell, or any combination thereof; and (ii) proliferationof a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any combination thereof. Insome embodiments, the immunosuppressive human myeloid cell is amacrophage. In some embodiments, the immunosuppressive human myeloidcell is a myeloid-derived suppressor cell. In some embodiments, theantibody is any embodiment as disclosed herein.

Disclosed herein, in certain embodiments, is a method of treating cancerin an individual in need thereof, the method comprising administering tothe individual a therapeutically effective amount of an antibody of anyone of the embodiments disclosed herein, thereby treating the cancer inthe individual.

Disclosed herein, in certain embodiments, is a method of treating cancerin an individual in need thereof, the method comprising administering tothe individual a therapeutically effective amount of an antibody of anyone of the embodiments disclosed herein, whereby immunosuppression by atumor-associated macrophage in the individual is reduced.

Disclosed herein, in certain embodiments, is a method of treating cancerin an individual in need thereof, the method comprising administering tothe individual a therapeutically effective amount of an antibody of anyone of the embodiments disclosed herein, whereby T cell-mediated tumorcell killing in the individual is increased. In some embodiments, thecancer is a lung cancer or sarcoma. In some embodiments, the lung canceris a lung carcinoma or lung adenocarcinoma.

Disclosed herein, in certain embodiments, is a method of reducing atumor promoting activity of a tumor-associated macrophage in anindividual in need thereof, the method comprising administering to theindividual an amount of a pharmaceutical composition that is effectiveto modulate a CD4⁺ T cell activation, CD4⁺ T cell proliferation, CD8⁺ Tcell activation, CD8⁺ T cell proliferation, or any combination thereofin the tumor microenvironment. In some embodiments, the method furthercomprises promoting tumor cell killing in the tumor microenvironment.

Disclosed herein, in certain embodiments, is a method of promotinglymphocyte-mediated tumor cell killing in an individual in need thereof,the method comprising administering to the individual an effectiveamount of a pharmaceutical composition of any one of the embodimentsdisclosed herein.

Disclosed herein, in certain embodiments, is a method of modulating anactivity of a tumor-associated macrophage in a tumor microenvironment,the method comprising contacting the tumor-associated macrophage with anantibody that binds to a CD163-expressing human macrophage and comprisesat least one of the following effects: (a) the binding of the antibodyreduces expression of at least one marker on the macrophage, wherein theat least one marker is CD16, CD64, TLR2, or Siglec-15; (b) upon bindingof the antibody with the CD163 protein, the antibody is internalized bythe human macrophage; (c) the binding of the antibody is not cytotoxicto the macrophage; (d) the binding of the antibody promotes anactivation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any combinationthereof; (e) the binding of the antibody promotes a proliferation of aCD4⁺ T cell, CD8⁺ T cell, NK cell, or any combination thereof; and (f)the binding of the antibody promotes tumor cell killing in the tumormicroenvironment. In some embodiments, the binding results in: two ormore of (a) through (f); three or more of (a) through (f); four or moreof (a) through (f); five or more of (a) through (f); or all of (a)through (f). In some embodiments, the activation of a CD4⁺ T cell, CD8⁺T cell, NK cell, or any combination thereof is measured as increasedproduction of IFN-γ, TNF-α, perforin, or any combination thereof. Insome embodiments, the method occurs in a tumor microenvironment. In someembodiments, the method occurs in vivo. In some embodiments, binding ofthe antibody to the macrophage modulates an immune function of a cell ina tumor microenvironment. In some embodiments, binding of the antibodyto the macrophage promotes an anti-tumor immune function. In someembodiments, the antibody comprises a constant domain and the constantdomain binds to an Fc receptor. In some embodiments, the Fc receptor isexpressed on the macrophage. In some embodiments, the method furthercomprises internalizing the antibody by the human macrophage uponbinding with the CD163 protein. In some embodiments, binding to theCD163 protein is not cytotoxic to the human macrophage. In someembodiments, the method further comprises promoting expression of CD69,ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereof by CD4⁺ Tcells. In some embodiments, the method further comprises promotingexpression of ICOS, OX40, PD1, LAG3, CTLA4, or any combination thereofby CD8⁺ T cells. In some embodiments, the method comprises reducingimmunosuppression in a tumor microenvironment. In some embodiments, themethod comprises promoting cytotoxic lymphocyte-mediated killing ofcancer cells. In some embodiments, the method comprises promoting NKcell-mediated tumor cell killing. In some embodiments, the methodcomprises promoting expression of IL-2 by T cells. In some embodiments,the method comprises increasing CD4⁺ T cells, CD196⁻ T cells, CXCR3⁺ Tcells, CCR4⁻ T cells, or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings of which:

FIG. 1 shows the AB101 antibody binding to human MDSC populations.

FIG. 2 shows AB101 binds to CD163^(Hi) cells.

FIG. 3 shows AB101 binding to human M2c, M1, and M0 as compared toisotype controls.

FIG. 4 shows AB101 binding to human peripheral blood T cells, B cells,NKT cells, neutrophils, monocytes, and dendritic cells, in which theisotype control is shown in gray and the AB101 binding is shown inblack.

FIG. 5 shows no binding of AB101 to a panel of human primary cells,including small airway epithelial cells (SAEC), renal proximal tubuleepithelial cells (RPTEC), lung microvascular endothelial cells (HMVEC),umbilical vein endothelial cells (HUVEC), aortic smooth muscle cells(AOSMC), and keratinocytes.

FIG. 6A shows the top 20 targets for AB102 based on mass spectrometryanalysis of a sample after immunoprecipitation.

FIG. 6B shows the top cell surface targets for AB102 based on massspectrometry analysis of a sample after immunoprecipitation.

FIG. 6C shows the top cell surface targets for AB102 compared to ISO(isotype negative control) based on mass spectrometry analysis of AB102and ISO samples after immunoprecipitation.

FIG. 7 shows AB102 coimmunoprecipitates a distinct higher molecularweight glycoform of CD163.

FIG. 8 shows AB101, AB102, and the control CD163 antibody bind tohuCD163, while the isotype control showed no appreciable binding.

FIG. 9 shows neither AB101 nor the control anti-huCD163 antibody boundto the recombinant murine CD163 compared to a commercially availableanti-muCD163 antibody, which did bind to murine CD163.

FIG. 10 shows pretreatment of M2c macrophages with polyclonal anti-CD163antibody blocked binding of the AB101 antibody compared to treatmentwith goat control polyclonal antibody, which did not block binding ofAB101.

FIG. 11 shows pretreatment of M2c macrophages with polyclonal anti-CD163antibody blocked binding of a control monoclonal anti-huCD163 antibodycompared to treatment with goat control polyclonal antibody, which didnot block binding of the control monoclonal anti-huCD163 antibody.

FIG. 12 shows treatment of polarized M2c macrophages with siRNA to CD163substantially reduced binding of the AB102 antibody compared to thescrambled siRNA (siScramb) or no siRNA treated M2c macrophages, and isrepresentative of the three replicates.

FIG. 13 shows siRNA knockdown of CD163 reduced binding of the AB102antibody, a slight decrease in AB102 antibody binding after knockdownwith siRNAs against FCGR2A+FCGR3A (in 1 out 3 donors), FCGR2C, orFCGR3A, and no evidence of reduction in AB102 binding after knockdownwith siCD206; siCD163L1; siPPIA; siLGALS1; siLGALS3; siLILRB2; andsiUPAR.

FIG. 14 shows treatment of the cultured M2 macrophages with LPS resultedin a loss of binding by both AB101 antibody and control anti-CD163antibody.

FIG. 15 shows increased IL-2 production after treatment of myeloid cellswith AB101 antibody.

FIG. 16 shows AB101 antibody treatment during polarization promoted CD4⁺T cell proliferation.

FIG. 17 shows AB101 antibody treatment during polarization promoted CD8⁺T cell proliferation.

FIG. 18 shows treatment with AB101 antibody during polarization (labeled“pre” on graph), post-polarization (labeled “post” on graph), orcombined during and post-polarization (labeled “pre and post” on graph)resulted in enhanced IL-2 production when compared to isotype antibodytreatment.

FIG. 19 shows treatment of M2 macrophages with the AB101 antibodyreduced expression of CD16, CD64, Calreticulin, and Siglec-15.

FIG. 20 shows that treatment of M2c cells with AB101 increased theTh1/Th2 ratio compared to the isotype control.

FIG. 21 shows that treatment of M2c cells with AB101 increased theexpression of CD69 on CD4 T cells compared to the isotype control.

FIG. 22 show that treatment of M2c cells with AB101 increased theexpression of ICOS on CD4 T cells compared to the isotype control.

FIG. 23 show that treatment of M2c cells with AB101 increased theexpression of OX40 on CD4 T cells compared to the isotype control.

FIG. 24 shows increased CTLs in the presence of BiTE resulted inincreased Raji tumor cell killing, compared to isotype control.

FIG. 25 shows the AB102 antibody internalized approximately as well asthe commercial anti-CD163 antibody (R&D Systems MAB1607-100), andapproximately 2-fold more of the AB101 antibody was internalized thanthat of the commercial CD163 antibody

FIG. 26 shows tumor volume plotted for the A549 tumors over 30 days.Arrows indicate injections with antibody treatments. Each pointrepresents the mean measurement from 7 mice. Error bars denote standarderror of the mean (SEM). Statistical significance was calculated usingMann-Whitney test.

FIG. 27 shows tumor volume plotted for the H1975 tumors over 30 days.Arrows indicate injections with antibody treatments. Each pointrepresents the mean measurement from 7 mice. Error bars denote standarderror of the mean (SEM). Statistical significance was calculated usingMann-Whitney test.

FIG. 28 shows the experimental design for M2c/T cell coculture assay toevaluate the effect of AB101 treatment on T cell proliferation and IL-2production.

FIG. 29 shows that treatment with AB101 during M2c macrophagepolarization restored T-cell proliferation in M2c/T cell cocultureassay.

FIG. 30 shows that treatment with AB101 during M2c macrophagepolarization enhanced IL-2 secretion by OKT3 activated T cells in M2c/Tcell coculture assay.

FIG. 31 shows that treatment with AB101 pre-, pre/post-, andpost-regimens increased CD8⁺ T cell proliferation in M2c/T cellcoculture assay.

FIG. 32 shows that treatment with AB101 pre-, pre/post-, andpost-regimens increased CD8⁺ T cell proliferation in M2c/T cellcoculture assay for individual subjects.

FIG. 33 shows that AB101 treatment enhanced CD8⁺ T cell proliferation inM2c/cocultures from multiple subjects.

FIG. 34 shows that AB101 treatment enhanced CD4⁺ T cell proliferation inM2c/cocultures from multiple subjects.

FIG. 35 shows that AB101 treatment enhanced IL-2 production by T cellsfrom multiple human during M2c/coculture.

FIG. 36 shows that AB101 is more potent than AB104 and AB102 isotypes inenhancing T cell proliferation in M2c/T cell coculture assay.

FIG. 37 shows that AB101, but not AB104, pre/post and post-regimenrescued CD8⁺ T cell proliferation from M2c mediated immune suppression.

FIG. 38 shows that AB101 restored CD8⁺ T cell cytokine response in M2c/Tcell coculture assay.

FIG. 39 shows that AB101 rescued CD4⁺ T cell IFN-γ, TNF-α and perforinresponse from M2c macrophage mediated immune suppression.

FIG. 40 shows that AB101 treatment enhanced the cytotoxic activity ofCD8⁺ T cells.

FIG. 41 shows that AB101 treatment enhanced the BiTE®-assisted cytotoxicactivity of CD8⁺ T cells.

FIG. 42 shows that AB101 treatment enhanced the cytotoxic activity ofnon-HLA restricted CD8⁺ T cells.

FIG. 43 shows that AB101 treatment relieved M2c cell mediated immunesuppression and induces a unique expression pattern by activated CD4⁺ Tcells.

FIG. 44 shows that AB101 treatment relieves M2c macrophages immunesuppression and enhances the activation of CD4⁺ and CD8⁺ T cells.

FIG. 45 shows CXCR3 expression by activated CD4⁺ T cells.

FIG. 46 shows that AB101 treatment during polarization of M2cmacrophages reduced the expression of CD16, CD64, Siglex-15 and TLR2 byM2c macrophages.

FIG. 47 shows a summary of changes based on pepsin digestion of AB101bound to huCD163. Figure discloses SEQ ID NO: 24.

FIG. 48 shows a summary of changes based on Nepenthesin II digestion ofAB101 bound to huCD163. Figure discloses SEQ ID NO: 24.

FIG. 49 shows schematic of AB101 binding to human CD163 ECD based onHDX-MS studies.

FIG. 50 shows that AB101 binds truncated CD163 ECD composed of SRCRdomain 1-5.

FIG. 51 shows alignment of human CD163 (SEQ ID NO:25) against cynomolgusCD163 (SEQ ID NO: 26). Signal sequence. The sequence under black barsindicate the 9 SRCR domains and the gray lines above the sequenceindicate consensus sequence. The protected and exposed regions are shownbased on AB101 binding epitope as determined by observed protection inHDX-MS. The solid bars under the sequence indicate a nepenthesin IIprotected region. The outlined box under the sequence indicates a pepsinprotected region. The hatched box under the sequence indicates a pepsinexposed region. The vertical striped box under the sequence indicates anepenthesin II exposed region. The lysine (K) at position 323 of humanCD163 and glutamic acid (E) of cynomolgus CD163 are indicated with abox.

FIG. 52 shows that AB101 binds human and cynomolgus E323K mutant butdoes not bind wildtype cynomolgus CD163 ECD.

FIG. 53 shows SPR detection of binding of AB101 to human CD163. AB101was serially diluted into different concentrations with (A) EDTA or (B)calcium-containing running buffer. GHI/61 was then injected into flowcell 2 with a flow rate at 30 μl/min, concentrations at6.25/12.5/25/50/100/200 μg/ml, a contact time of 300 s, and adissociation time of 600 s.

FIG. 54 shows SPR detection of binding of GHI/61 to human CD163.Anti-CD163 clone GHI/61 was serially diluted into differentconcentrations with (A) EDTA or (B) calcium-containing running buffer.GHI/61 was then injected into flow cell 2 with a flow rate at 30 μl/min,concentrations at 3.125/6.25/12.5/25/50/100 μg/ml, a contact time of 300s, and a dissociation time of 600 s.

FIG. 55 shows SPR detection of binding of CD163 to AB101. Human CD163protein was serially diluted into different concentrations withcalcium-containing running buffer. CD163 protein was then injected intoflow cell 2 with a flow rate at 30 μl/min, concentrations at1.25/2.5/5.0/10.0/20.0/40.0 μg/ml, a contact time of 300 s, and adissociation time of 600 s.

FIG. 56 shows binding of AB101 to soluble CD163 in AlphaLisa assay.AB101 (circles) or isotype control (triangles) were incubated with 750nM CD163-His at the indicated concentration for 1 h. Binding wasquantified by AlphaLisa with a biotinylated anti-hIgG1 mAb, streptavidinacceptor and nickel donor beads. Symbols represent the mean±standarderror of five independent measurements. Curve fit was performed with 1and 2-site saturated binding models (GraphPad Prism). (R²=0.92). (A)linear and (B) log x-axes scale.

FIG. 57 shows AB101 binding to M1c macrophages. M2c macrophages wereblocked with stringent FACS blocking buffer containing 0.5 mg/ml humanIgG1 and then stained with AB101 (circles) or isotype control(triangles) at the indicated concentration for 30 min. Binding ofAlexaFluor 647 labeled AB101 and isotype control to M2c macrophages wasquantified by fluorescence intensity and reported as gMFI. Symbolsrepresent the mean±standard error of four study subjects. Curve fits wasperformed with the 2-site saturated binding model (GraphPad Prism).(R²=0.99). (A) linear and (B) log x-axes scale.

DETAILED DESCRIPTION OF THE DISCLOSURE

Disclosed herein are antibodies that specifically bind to CD163⁺ cells.In some embodiments, the CD163⁺ cells are immunosuppressive myeloidcells. In some embodiments, the CD163⁺ cells are human CD163 expressingmyeloid. In some embodiments, the CD163⁺ cells are tumor cells. In someembodiments, the CD163⁺ immunosuppressive myeloid cells are humanmacrophages. In some embodiments, the human CD163⁺ immunosuppressivemacrophages are M2 or M2-like macrophages. In some embodiments, theimmunosuppressive myeloid cells are myeloid-derived suppressor cells(MDSC). In some embodiments, the human macrophages express high levelsof CD163 (CD163^(Hi)). By contrast, other human hematopoietic cells orprimary non-immune human cells do not express CD163. For example, M1 andM1-like macrophages do not express CD163.

Monocytes and macrophages exposed to certain inflammatory cytokines ormicrobe-associated molecular patterns differentiate intopro-inflammatory (M1 or M1-like) or anti-inflammatory M2 or M2-likemacrophages. M1 and M2 are classifications used to define macrophagesactivated in vitro as pro-inflammatory (when classically activated withIFN-γ and lipopolysaccharide) or anti-inflammatory (when alternativelyactivated with IL-4 or IL-10), respectively, whereas in vivo or ex vivomacrophages with M1 or M2 phenotypes are defined as M1-like or M2-likemacrophages. In some embodiments, M2 macrophages are generated by theirexposure to certain cytokines. In some embodiments, the M2 macrophagesare differentiated by IL-4, IL-10, IL-13, or a combination thereof.

Sub-types of M2 macrophages include M2a, M2b, M2c, and M2d subtypes. M2amacrophages are induced by IL-4 and IL-13 which evokes unregulatedexpression of CD163, arginase-1, mannose receptor MRC1 (CD206), antigenpresentation by MHC II system, and production of IL-10 and TGF-β,leading to tissue regeneration and the inhibition of pro-inflammatorymolecules to prevent the inflammatory response. The M2b macrophagesproduce IL-1, IL-6, IL-10, TNF-α as a response to immune complexes. M2cmacrophages are induced by IL-10, transforming growth factor beta(TGF-β) and glucocorticoids exposure, and produce IL-10 and TGF-β,leading to suppression of inflammatory response. M2d subtypes areactivated as a response to IL-6 and adenosines.

M2 macrophages have functions and phenotypes corresponding to M2macrophages and their subtypes. An M2-like macrophage is any in vivo orex vivo macrophage having a subset of the functional or phenotypiccharacteristics of M2 macrophages.

In some embodiments, the antibodies of the present disclosure have highavidity and specific binding for immunosuppressive myeloid cells, inparticular, for tumor-associated macrophages, such as M2 and M2-likemacrophages. In some embodiments, the antibodies specifically bind to M2and M2-like TAMs from human primary lung tumors. In some embodiments,the antibodies as disclosed herein do not have appreciable binding to M1or M1-like macrophages. M1-activated macrophages express transcriptionfactors such as Interferon-Regulatory Factor (IRF5), Nuclear Factor ofkappa light polypeptide gene enhancer (NF-κB), Activator-Protein (AP-1)and STAT1. M1 macrophages secrete pro-inflammatory cytokines such asIFN-γ, IL-1, IL-6, IL-12, IL-23 and TNFα. M1 macrophages have functionsand phenotypes corresponding to M1 macrophages. An M1-like macrophage isany in vivo or ex vivo macrophage having a subset of the functional orphenotypic characteristics of M1 macrophages.

In some embodiments, the antibodies of the present disclosure do notbind to primary human cells. In some embodiments, the antibodies of thepresent disclosure do not bind to hematopoietic stem cells, leukocytes,T cells, B cells, NK cells, and granulocytes.

Tumor-associated macrophages (TAMs) are a heterogeneous class ofmacrophage cells present in high numbers in the microenvironment ofsolid tumors. Most evidence suggests that TAMs have a tumor-promotingphenotype, appearing to be involved in tumor cell proliferation, tumorangiogenesis, motility, and invasion, metastasis, anticancer drugresistance, and tumor immune evasions.

Direct tumor cell killing by cytotoxic T cells among tumor-infiltratinglymphocytes (TIL) play a major role in the anti-tumor function of theimmune system. TAMs in the tumor microenvironment (TME), however,suppress the T cell-mediated anti-tumor immune response. TAMs have animmunosuppressive transcriptional profile and express factors includingIL-10 and transforming growth factor β (TGFβ). In humans, TAMs have beenshown to directly suppress T cell function through surface presentationof programmed death-ligand 1 (PD-L1) in hepatocellular carcinoma andB7-homologs in ovarian carcinoma, which activate programmed cell deathprotein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4(CTLA4), respectively, on T cells. Inhibitory signals to PD-1 and CTLA4are immune checkpoints, and binding of these inhibitory receptors bytheir ligands inhibits T cell receptor signaling and T cell cytotoxicfunction, and promotes T cell apoptosis. HIF-1α induces TAMs to suppressT cell function. CD163 has been identified as an immunosuppressivemolecule that is solely expressed on TAMs, and could be a potentialtherapeutic target for cancer immunotherapy.

TAMs generally fall into two categories, M1-like antitumor and M2-likeimmunosuppressive macrophages, based on their functionalcharacteristics, including their relationships to T helper cell (CD4⁺)types Th1 and Th2. M1 macrophages are a model of “classical” and can begenerated with IFN-γ with either innate immune activators such aspathogen associated molecular patters (PAMP) (e.g., lipopolysaccharide(LPS)) or damage-associated molecular patterns (DAMPs) as well asinflammatory cytokines (e.g., tumor necrosis factor-alpha (TNF-α). Inaddition, T cell dependent macrophage activation via the CD40-CD40ligand pathway induce M1 differentiation. M1 macrophages havepro-inflammatory, bactericidal, and cytotoxic functions. Thesemacrophages promote the antigen-dependent induction of Th1 cells andactivation of Th1 and CD8⁺ T cells. The promotion of T cell cytotoxicactivity by M1-like anti-tumor macrophages is critical for tumor cellelimination. In some embodiments, M1-like anti-tumor macrophages arecharacterized by surface marker expression measured by flow cytometryand have either a CD80⁺ CD86⁺ CD163^(Lo/−) or CD206^(Lo/−) phenotype. M1macrophages secrete IL-12, and low level of IL-10 and/or TGF-β.

By contrast, M2-like immunosuppressive macrophages are a model of“alternative” or “non-classical” activation, which can be generated withIL-4 or IL-10 in vitro, are anti-inflammatory and promote wound healingand tissue repair. In some embodiments, M2-like immunosuppressivemacrophages are polarized from monocyte-derived macrophages andrecruited by factors secreted by tumors. M2-like immunosuppressivemacrophages are the principal macrophage cell type involved inpro-tumoral function of TAMs, which includes promoting tumor growth,metastasis, and immune evasion. M2-like macrophages express the surfacemarkers CD15, CD23, CD64, CD68, CD163^(Hi), CD204^(Hi), CD206^(Hi),and/or other M2 macrophage markers determined by flow cytometry. M2macrophages secrete high levels of IL-10 and TGF-beta1, and low levelsof IL-12.

In many tumor types, TAM infiltration level has significant prognosticvalue. TAMs have been linked to poor prognosis in a wide variety oftumors. For example, it has been found that breast cancer patients withmore M2-like tumor-associated macrophages had higher-grade tumors,greater microvessel density, and lower overall survival. Patients withmore M1-like anti-tumor TAMs displayed the opposite effect.

Accordingly, there remains a need to identify compounds and methods toimprove immunotherapeutic treatment of treating cancer.

CD163 (scavenger receptor cysteine-rich type 1 protein M130; hemoglobinscavenger receptor) is a cell surface protein which acts as a scavengerreceptor for the hemoglobin-haptoglobin complex and protects tissuesfrom free hemoglobin-mediated oxidative damage. Four isoforms of CD163protein, with molecular weights of 125,451, 125,982, 121,609 and 124,958Da have been reported. Isoform 1 is the most prevalent isoform of CD163,with a molecular weight of 125,451 Da, and consisting of 1115 aminoacid-residue polypeptide comprising an extracellular domain, atransmembrane segment, and a cytoplasmic tail. The extracellular domaincomprises nine cysteine-rich repeat domains. Isoform 1 of CD163 proteinhas four N-linked glycosylation sites, and in M2 macrophages CD163protein shows two distinct bands, at ˜150 kDa and ˜130 kDa, in SDS-PAGEunder reducing conditions.

CD163 mRNA expression is generally restricted to myeloid cells but isalso expressed by certain human cancers. CD163 expression on TAMs hasbeen associated with immunosuppressive M2-like phenotype, which has beenshown to correlate with poor clinical outcome in cancer. CD163 isrequired for protumoral activation of macrophages in human and murinesarcoma. CD163 has also been reported to be a macrophage scavengerreceptor and promote immunosuppression. In some embodiments, theinteraction of the hemoglobin-haptoglobin complex with CD163 induces thesecretion of the immunosuppressive cytokine IL-10 and the expressionheme-oxygenase-1 (HO-1). HO-1 produces the anti-inflammatory metabolitesFe²⁺, CO and biliverdin.

Soluble CD163 occurs in humans via ectodomain shedding and is reportedto have anti-inflammatory properties, such as downregulating T-cellresponses, including lymphocyte proliferation stimulated byphytohemagglutinin (PHA) or 12-O-tetradecanoylphorbol-13-acetate (TPA).

Antibodies targeting CD163 have been shown to modulate the innate immuneresponse of CD163 expressing macrophages. For example, RM3/1 antibody,an antibody against CD163, is a mouse monoclonal IgG1 (kappa lightchain) that was raised against human monocytes. The RM3/1 antibody bindsto the cysteine-rich domain 9 of human CD163, reduces LPS-induced TNFα,and enhances IL-10 secretion by macrophages.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. Generally,nomenclatures utilized in connection with, and techniques of,immunology, oncology, cell and tissue culture, molecular biology, andprotein and oligo- or polynucleotide chemistry and hybridizationdescribed herein are those well-known and commonly used in the art. Itis to be understood that the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of any subject matter claimed. The section headingsused herein are for organizational purposes only and are not to beconstrued as limiting the subject matter described.

As used herein, singular forms “a,” “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “an antibody” includes a plurality of antibodiesand reference to “an antibody” in some embodiments includes multipleantibodies, and so forth.

As used herein, all numerical values or numerical ranges include wholeintegers within or encompassing such ranges and fractions of the valuesor the integers within or encompassing ranges unless the context clearlyindicates otherwise. Thus, for example, reference to a range of 90-100%,includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%,91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%,etc., and so forth. In another example, reference to a range of 1-5,000fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5 fold, etc.,2.1, 2.2, 2.3, 2.4, 2.5 fold, etc., and so forth.

“About” a number, as used herein, refers to range including the numberand ranging from 10% below that number to 10% above that number. “About”a range refers to 10% below the lower limit of the range, spanning to10% above the upper limit of the range.

“Percent identity” and “% identity” refers to the extent to which twosequences (nucleotide or amino acid) have the same residue at the samepositions in an alignment. For example, “an amino acid sequence is X %identical to SEQ ID NO: Y” refers to % identity of the amino acidsequence to SEQ ID NO:Y and is elaborated as X % of residues in theamino acid sequence are identical to the residues of sequence disclosedin SEQ ID NO: Y. Generally, computer programs are employed for suchcalculations. Exemplary programs that compare and align pairs ofsequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988March; 4(1):11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci USA.1988 April; 85(8):2444-8; Pearson, Methods Enzymol. 1990; 183:63-98) andgapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep. 1;25(17):3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic AcidsRes. 1984 Jan. 11; 12(1 Pt 1):387-95).

As used herein “antibody” refers to a protein that binds an antigen. Anantibody often comprises a variable domain and a constant domain in eachof a heavy chain and a light chain. Accordingly, most antibodies have aheavy chain variable domain (VH) and a light chain variable domain (VL)that together form the portion of the antibody that binds to theantigen. Within each variable domain are threecomplementarity-determining regions (CDR), which form loops in the heavychain variable domain (VH) and light chain variable domain (VL) andcontact the surface of the antigen. “Antibody” includes, but is notlimited to, polyclonal, monoclonal, monospecific, multispecific (e.g.,bispecific antibodies), natural, humanized, human, chimeric, synthetic,recombinant, hybrid, mutated, grafted, antibody fragments (e.g., aportion of a full-length antibody, generally the antigen binding orvariable region thereof, e.g., Fab, Fab′, F(ab′)2, and Fv fragments),and in vitro-generated antibodies having the antigen-binding activity.The term also includes single chain antibodies, e.g., single chain Fv(sFv or scFv) antibodies, in which a variable heavy and a variable lightchain are joined together (directly or through a peptide linker) to forma continuous polypeptide.

As used herein “complementarity-determining regions (CDRs)” refers tothe part of the variable chains in antibodies that bind to the specificantigen. Multiple methods may be used to define a CDR. The current artutilizes various numbering schemes with different definitions of CDRlengths and positions. For example, the Kabat numbering scheme is basedon sequence alignment and uses “variability parameter” of a given aminoacid position (the number of different amino acids at a given positiondivided by the frequency of the most occurring amino acid at thatposition) to predict CDRs. The Chothia numbering scheme, on the otherhand, is a structure-based numbering scheme where antibody crystalstructures are aligned as define the loop structures as CDRs. The Martinnumbering scheme focuses on the structure alignment of differentframework regions of unconventional lengths. IMGT numbering scheme is astandardized numbering system based on alignments of sequences from acomplete reference gene database including the whole immunoglobulinsuperfamily. Honneger's numbering scheme (AHo's) is based on structuralalignments of the 3D structure of the variable regions and usesstructurally conserved Ca positions to deduce framework and CDR lengths.One of skill in the art will note that the definition of a CDR will varybased on the method used. Any method of defining a CDR is contemplatedwith the sequences disclosed herein.

The terms “recipient,” “individual,” “subject,” “host,” and “patient,”are used interchangeably herein and refer to any mammalian subject forwhom diagnosis, treatment, or therapy is desired, particularly humans.“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and laboratory,zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep,goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc. In someembodiments, the mammal is a human.

As used herein, the terms “treatment,” “treating,” and the like, in somecases, refer to administering an agent, or carrying out a procedure, forthe purposes of obtaining an effect. In some embodiments, the effect isprophylactic in terms of completely or partially preventing a disease orsymptom thereof and/or is therapeutic in terms of effecting a partial orcomplete cure for a disease and/or symptoms of the disease. “Treatment,”as used herein, includes treatment of a disease or disorder (e.g.,cancer) in a mammal, particularly in a human, and includes: (a)preventing the disease or a symptom of a disease from occurring in asubject which is predisposed to the disease but has not yet beendiagnosed as having it (e.g., including diseases that is associated withor caused by a primary disease; (b) inhibiting the disease, i.e.,arresting its development; and (c) relieving the disease, i.e., causingregression of the disease. In some embodiments, treating refers to anyindicia of success in the treatment or amelioration or prevention of acancer, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the diseasecondition more tolerable to the patient; slowing in the rate ofdegeneration or decline; or making the final point of degeneration lessdebilitating. The treatment or amelioration of symptoms is based on oneor more objective or subjective parameters; including the results of anexamination by a physician. Accordingly, the term “treating” includesthe administration of the compounds or agents of the present disclosureto prevent or delay, to alleviate, or to arrest or inhibit developmentof the symptoms or conditions associated with diseases (e.g., cancer).The term “therapeutic effect” refers to the reduction, elimination, orprevention of the disease, symptoms of the disease, or side effects ofthe disease in the subject. A subject is “treated” for a disease ordisorder if, after receiving a therapeutic amount of an antibody of thepresent disclosure, the patient shows observable and/or measurablechange in a parameter or symptom of the disease or disorder, such as, inthe case of treatment of a cancer, increased tumor cell killing activityas assessed ex vivo, lower levels of immunosuppressive secreted factorsin blood, lower tumor volume or mass, increased cytotoxic lymphocytesand Th1 like T cell numbers in tumor biopsy, reduced morbidity ormortality, improvement in quality of life factors, or improvement in anyobjective indicia related to a parameter or symptom of the disease ordisorder. In some embodiments, the parameters include converting immunecold tumors into immune hot, e.g., by increasing cytotoxic lymphocytecell number as well as markers of T cell activation (CD69, ICOS, OX40,etc.) in tumor biopsy, or decreasing expression of CD16, CD64, TLR2,Siglec-15 on TAMs in tumor biopsy.

In some embodiments, “inducing a response” refers to the alleviation orreduction of signs or symptoms of illness in a subject, and specificallyincludes, without limitation, prolongation of survival.

The term “avidity” refers to the resistance of a complex of two or moreagents to dissociation after dilution.

In some embodiments, antibody “effector functions” refers to thosebiological activities attributable to the Fc region (a native sequenceFc region or amino acid sequence variant Fc region) of an antibody andvary with the antibody isotype.

“Fc receptor” or “FcR” refers to a receptor that binds to the Fc regionof an antibody.

“Human effector cells” as used herein refers to leukocytes that expressone or more FcRs and perform effector functions. For example, the cellsexpress at least FcγRIII and perform an ADCC effector function. Examplesof human leukocytes that mediate ADCC include, but are not limited to,peripheral blood mononuclear cells (PBMC), NK cells, monocytes,macrophages, cytotoxic T cells, and neutrophils.

“Complement-dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)that are bound to their cognate antigen. To assess complementactivation, a CDC assay, for example, is performed.

An antibody that “internalizes” is one that is taken up by (i.e.,enters) the cell upon binding to an antigen on a mammalian cell (e.g., acell surface polypeptide or receptor). The internalizing antibodycomprises antibody fragments, human or chimeric antibody, and antibodyconjugates. In some cases, internalization of an antibody (e.g., such asdisclosed herein) alter the biology of the cell, causing it to changeits function.

An “antigen-binding domain,” “antigen-binding region,” or“antigen-binding site” is a portion of an antibody that contains aminoacid residues (or other moieties) that interact with an antigen andcontribute to the antibody's specificity and affinity for the antigen.For an antibody that specifically binds to its antigen, this willinclude at least part of at least one of its CDR domains.

The antigen-binding region of an antibody is referred to as a“paratope,” which binds to an antigenic determinant, the “epitope” of anantigen, that is, a portion of the antigen molecule that is able to bebound by an antibody. In some embodiments, an antigen substance has oneor more portions that are recognizable by antibodies, i.e., more thanone epitope, and thus a single antigen substance is specifically boundby different antibodies each having specificity for a different epitope.In some embodiments, an epitope comprises non-contiguous portions of theantigen. For example, in a polypeptide, amino acid residues that are notcontiguous in the polypeptide's primary sequence but that, in thecontext of the polypeptide's tertiary and quaternary structure, are nearenough to each other to be bound by an antigen-binding protein,constitutes an epitope.

An “antibody fragment” comprises a portion of an intact antibody. Insome embodiments, the antibody fragment comprises an antigen-binding orvariable region of the intact antibody.

The terms “antigen-binding portion of an antibody,” “antigen-bindingfragment,” “antigen-binding domain,” “antibody fragment” are usedinterchangeably herein to refer to one or more fragments of an antibodythat retain the ability to specifically bind to the antigen.Non-limiting examples of antibody fragments included within such termsinclude, but are not limited to, (i) a Fab fragment, a monovalentfragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab′)2fragment, a bivalent fragment containing two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment consisting ofthe VH and CH domains; (iv) a Fv fragment containing the VL and VHdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,Nature 341(6242):544-6 (1989)), containing a VH domain; and (vi) anisolated CDR. Also included are “one-half” antibodies comprising asingle heavy chain and a single light chain. Other forms of single chainantibodies, such as diabodies are also encompassed herein.

A “functional antibody fragment” as used herein refers in context to anantibody fragment that not only binds the antibody's antigen, but alsopossesses a functional attribute that characterizes the intact antibody.For example, if an antibody depends for a function on possessing a Fcdomain that enables an effector function, such as ADCC, a functionalfragment would possess such function. It is hypothesized that antibodiesof the disclosure are effective in modulating the functional state oftumor-associated macrophages, or reorienting or dampening the M2-statusmacrophages, when they comprise an Fc portion that binds to a macrophageFc receptor, such as CD16 (FcγRIIIa) or CD64 (FcγRI) in someembodiments.

The phrase “functional fragment or analog” of an antibody is a compoundhaving qualitative biological activity in common with a full-lengthantibody. For example, a functional fragment or analog of an anti-IgEantibody is one that binds to an IgE immunoglobulin to prevent orsubstantially reduce the ability of such molecule from having theability to bind to the high affinity receptor, FcγRI.

An “antigen-binding protein” is a protein comprising a portion thatcomprises an antigen-binding portion of an antibody, optionally alsoincluding a scaffold or framework portion that allows theantigen-binding portion to adopt a conformation that promotes binding ofthe antigen-binding protein to the antigen.

An “intact” antibody is one that comprises an antigen-binding site aswell as a C_(L) and at least heavy chain constant domains, C_(H)1,C_(H)2, and C_(H)3. In some embodiments, the constant domains are nativesequence constant domains (e.g., human native sequence constant domains)or amino acid sequence variant thereof.

The term “recombinant antibody” as used herein refers to an antibodycomprising an antigen-binding domain of a first antibody, such as, forexample, a CDR, a VH region, or an intact light chain, and a domain fromone or more other antibodies or proteins. Chimeric, hybrid, andhumanized antibodies are examples of recombinant antibodies.

A “CDR-grafted antibody” is an antibody comprising one or more CDRsderived from an antibody of one species or isotype and the framework ofanother antibody of the same or different species or isotype.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In one embodiment, all of the variable and constant domainsof the antibody are derived from human immunoglobulin sequences(referred to as a “fully human antibody”).

As used herein, the term “affinity” refers to the equilibrium constantfor the reversible binding of two agents and is expressed as K_(D). Inone embodiment, the antibodies or antigen-binding fragments thereofexhibit binding affinity as measured by K_(D) (equilibrium dissociationconstant) for CD163 in the range of 10⁻⁶ M or less, or ranging down to10⁻¹⁶M or lower, (e.g., about 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹²,10⁻¹³, 10⁻¹⁴, 10⁻¹⁵, 10⁻¹⁶M or less). In certain embodiments, antibodiesas describe herein specifically bind to a huCD163 polypeptide with aK_(D) of less than or equal to 10⁻⁴M, less than or equal to about 10⁻⁵M, less than or equal to about 10⁻⁶ M, less than or equal to 10⁻⁷M, orless than or equal to 10⁻⁸M.

The terms “preferentially binds” or “specifically binds” mean that theantibodies or fragments thereof bind to an epitope with greater affinitythan it binds unrelated amino acid sequences, and, if cross-reactive toother polypeptides containing the epitope, are not toxic at the levelsat which they are formulated for administration to human use. In someembodiments, such affinity is at least 1-fold greater, at least 2-foldgreater, at least 3-fold greater, at least 4-fold greater, at least5-fold greater, at least 6-fold greater, at least 7-fold greater, atleast 8-fold greater, at least 9-fold greater, 10-fold greater, at least20-fold greater, at least 30-fold greater, at least 40-fold greater, atleast 50-fold greater, at least 60-fold greater, at least 70-foldgreater, at least 80-fold greater, at least 90-fold greater, at least100-fold greater, or at least 1000-fold greater than the affinity of theantibody or fragment thereof for unrelated amino acid sequences.

The term “specific” refers to a situation in which an antibody willpreferentially bind to molecules other than the antigen containing theepitope recognized by the antibody. The term is also applicable wherefor example, an antigen-binding domain is specific for a particularepitope which is carried by a number of antigens, in which case theantibody or antigen-binding fragment thereof carrying theantigen-binding domain will be able to bind to the various antigenscarrying the epitope.

As used herein, an antibody is said to be “immunospecific” or “specific”for, or to “specifically bind” to, an antigen if that antibody reacts ata detectable level with the antigen, preferably with an affinityconstant, K_(a), of greater than or equal to about 10⁴M⁻¹, or greaterthan or equal to about 10⁵ M⁻¹, greater than or equal to about 10⁶M⁻¹,greater than or equal to about 10⁷ M⁻¹, or greater than or equal to 10⁹M⁻¹.

The term “monospecific,” as used herein, refers to an antibodycomposition that contains an antibody that displays a preferentialaffinity for one particular epitope. In some embodiments, monospecificantibody preparations are made up of about 10%, 20%, 30%, 40%, 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% antibody havingspecific binding activity for the particular antigen.

The term “polypeptide” is used in its conventional meaning, i.e., as asequence of amino acids. The polypeptides are not limited to a specificlength of the product. Peptides, oligopeptides, and proteins areincluded within the definition of polypeptide, and such terms are usedinterchangeably herein unless specifically indicated otherwise. Thisterm also does not refer to or exclude post-expression modifications ofthe polypeptide, for example, glycosylations, acetylations,phosphorylations, and the like, as well as other modifications known inthe art, both naturally occurring and non-naturally occurring. In someembodiments, a polypeptide is an entire protein, or a subsequencethereof. Particular polypeptides of interest in the context of theantibodies of this disclosure are amino acid subsequences comprisingCDRs and being capable of binding human M2 macrophages or CD163 proteinexpressed by such cells.

As used herein, “substantially pure,” and “substantially free” refer toa solution or suspension containing less than, for example, about 20% orless extraneous material, about 10% or less extraneous material, about5% or less extraneous material, about 4% or less extraneous material,about 3% or less extraneous material, about 2% or less extraneousmaterial, or about 1% or less extraneous material.

The term “isolated” refers to a protein (e.g., an antibody), nucleicacid, or other substance that is substantially free of other cellularmaterial and/or chemicals. In some embodiments, the antibodies, orantigen-binding fragments thereof, and nucleic acids of the disclosureare isolated. In some embodiments, the antibodies, or antigen-bindingfragments thereof, and nucleic acids of the disclosure are substantiallypure.

When applied to polypeptides, “isolated” generally means a polypeptidethat has been separated from other proteins and nucleic acids with whichit naturally occurs. Preferably, the polypeptide is also separated fromsubstances such as antibodies or gel matrices (polyacrylamide) which areused to purify it. In some cases, the term means a polypeptide or aportion thereof which, by virtue of its origin or manipulation: (i) ispresent in a host cell as the expression product of a portion of anexpression vector; or (ii) is linked to a protein or other chemicalmoiety other than that to which it is linked in nature; or (iii) doesnot occur in nature, for example, a protein that is chemicallymanipulated by appending, or adding at least one hydrophobic moiety tothe protein so that the protein is in a form not found in nature. By“isolated” it is further meant a protein that is: (i) synthesizedchemically; or (ii) expressed in a host cell and purified away fromassociated and contaminating proteins.

The term “effective amount” as used herein, refers to that amount of anantibody, or an antigen-binding portion thereof as described herein,that is sufficient to induce a response, e.g., to effect treatment,prognosis, or diagnosis of a disease associated with macrophage activityor TAM activity, as described herein, when administered to a subject.Therapeutically effective amounts of antibodies provided herein, whenused alone or in combination, will vary depending upon the relativeactivity of the antibodies and combinations (e.g., in inhibiting tumorcell growth) and depending upon the subject and disease condition beingtreated, the weight and age of the subject, the severity of the diseasecondition, the manner of administration, and the like, which, in somecases, are readily determined by one of ordinary skill in the art.

The term “therapeutically effective amount” generally refers to anamount of an antibody or a drug effective to “treat” a disease ordisorder in a subject or mammal. In some embodiments, a compositiondescribed herein is administered to a subject in an amount that iseffective for producing some desired therapeutic effect by inhibiting adisease or disorder as described herein at a reasonable benefit/riskratio applicable to any medical treatment. A therapeutically effectiveamount is an amount that achieves at least partially a desiredtherapeutic or prophylactic effect in an organ or tissue. The amount ofan antibody necessary to bring about prevention and/or therapeutictreatment of a disease or disorder is not fixed per se. In someembodiments, the amount of the antibody administered varies with thetype of disease, extensiveness of the disease, and size of the mammalsuffering from the disease or disorder. When used in conjunction withtherapeutic methods involving administration of a therapeutic agentafter the subject presents symptoms of a disease or disorder, the term“therapeutically effective” means that, after treatment, one or moresigns or symptoms of the disease or disorder is ameliorated oreliminated.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness, and the like, when administered to a human.

The term “contacting” is defined herein as a means of bringing acomposition as provided herein in physical proximity with a cell, organ,tissue, or fluid as described herein.

Immunomodulatory Antibodies

Disclosed herein, in certain embodiments, are antibodies thatspecifically bind to a CD163 protein expressed on human CD163⁺ cell. Insome embodiments, the CD163⁺ cell is an immunosuppressive myeloid cell.In some embodiments, the immunosuppressive myeloid cell is humanmacrophage. In some embodiments, the binding of an antibody disclosedherein alters expression of at least one marker on the human macrophage.

In some embodiments, an antibody disclosed herein binds to a human CD163(huCD163) protein expressed on a human M2 or M2-like immunosuppressivemacrophage. In some embodiments, the antibody specifically binds to aCD163 protein that is an approximately 140 kDa glycoform of huCD163. Insome embodiments, the antibody specifically binds to extracellulardomain 3 of huCD163. In some embodiments, the antibody specificallybinds to extracellular domain 4 of huCD163. In some embodiments, theantibody specifically binds to extracellular domain 3 and extracellulardomain 4 of huCD163. In some embodiments, the antibody specificallybinds to huCD163, resulting in a conformational change of huCD163. Insome embodiments, the conformational change to humCD163 exposesextracellular domains 2, 5, and 9 of humCD163. In some embodiments, theantibody does not specifically bind a lower molecular weight(approximately 115 kDa) glycoform of huCD163.

In some embodiments, an antibody disclosed herein binds to a humanCD163⁺ immunosuppressive myeloid cell and causes an alteration in theexpression of certain cell markers that characterize a M2 or M2-likeimmunosuppressive macrophage (such as a M2c macrophage), indicating afunctional differentiation of the macrophages to a non- or lessimmunosuppressive as well as a more anti-tumor state. In someembodiments, an antibody disclosed herein binds to a M2 or M2-likeimmunosuppressive macrophage and causes a decrease in the expression ofcertain cell markers that characterize a M2 or M2-like macrophage,indicating a functional differentiation of the macrophages to an altereddifferentiation state. In some embodiments, an antibody disclosed hereinreduces expression of one or more of CD16, CD64, TLR2, and Siglec-15 bythe CD163⁺ immunosuppressive myeloid cell.

In some embodiments, the binding of an antibody disclosed herein to aCD163⁺ immunosuppressive myeloid cells results in a functional change inthe CD163⁺ immunosuppressive myeloid cell. In some embodiments, thebinding of the antibody disclosed herein to the CD163⁺ immunosuppressivemyeloid cell results in changes in marker expression in the M2 orM2-like immunosuppressive macrophages. In some embodiments, thefunctional changes in the antibody-bound CD163⁺ immunosuppressivemyeloid cells result in modified interactions with other cells, such aseffector T cells, which subsequently modifies their effects on andinteraction with target tumor cells.

In some embodiments, an antibody of the present disclosure reducesimmunosuppression caused by tumor-associated macrophages in tumormicroenvironments. In some embodiments, a reduction in immunosuppressionby tumor-associated macrophages in the tumor microenvironmentcorresponds to an increase in immunostimulation, e.g., production ofpromotion of T cell activation, T cell proliferation, NK cellactivation, NK cell proliferation, or any combination thereof. In someembodiments, T cell activation and/or NK cell activation results inincreased production of IFN-γ, TNF-α, perforin, or a combination thereofby T cells and/or NK cells. In some embodiments, the antibodies of thepresent disclosure increase immunostimulation, e.g., production ofpromotion of T cell activation, T cell proliferation, NK cellactivation, NK cell proliferation, or any combination thereof. In someembodiments, T cell activation and/or NK cell activation results inincreased production of IFN-γ, TNF-α, perforin, or a combination thereofby T cells and/or NK cells. In some embodiments, antibodies of thepresent disclosure specifically bind to a CD163 protein expressed on ahuman macrophage, wherein the human macrophage has a firstimmunosuppression activity before binding the antibody and secondimmunosuppression activity after binding the antibody, and wherein thesecond immunosuppression activity lower than the first immunosuppressionactivity. In various embodiments, the first and second immunosuppressionactivities are each non-zero.

In some embodiments, an antibody of the present disclosure promotes Tcell activation and proliferation. In some embodiments, the antibodyskews a T cell population towards an anti-tumor T cell phenotype. Insome embodiments, the antibody reduces or blocks myeloid cellsuppression of T cell activation. In some embodiments, the antibodyreduces the ability of TAMs to suppress T-cell activation, leading togreater T-cell stimulation and IL-2 production. In some embodiments, theantibody blocks the ability of TAMs to suppress T-cell activation,leading to greater T-cell stimulation and IL-2 production.

In some embodiments, an antibody disclosed herein reduces myeloidsuppression of T cell proliferation. In some embodiments, the antibodyreduces the ability of TAMs to suppress both CD4⁺ and CD8⁺ T cellactivation and proliferation. In some embodiments, the antibody reducesTAM suppression of Th1 cell proliferation. Proliferated T cells showenhanced expression of activation markers on CD4⁺ T cells.

In some embodiments, an antibody of the present disclosure alters anM2-polarized macrophage such that the macrophage exhibits a M1-likephenotype that alleviates immunosuppressive effects of M2 macrophages.In some embodiments, an antibody described herein influencesmonocyte-derived macrophages to differentiate to a lessimmunosuppressive and more anti-tumor differentiation state.

In some embodiments, provided herein are antibodies that specificallybind to a human CD163 protein (huCD163) that is expressed on a humanmacrophage and reduces expression of at least one of CD16, CD64, TLR2,or Siglec-15 by the macrophage. In some embodiments, the humanmacrophage is tumor-associated immunosuppressive macrophage. In someembodiments, the human macrophage is an M2-like immunosuppressivemacrophage.

In some embodiments, an antibody disclosed herein binds to a CD163protein that is expressed by a macrophage as a component of a complexcomprising at least one other protein expressed by the macrophages. Insome embodiments, the complex is a cell surface complex. In someembodiments, the complex comprises at least one other protein selectedfrom a galectin-1 protein, a LILRB2 protein, and a casein kinase IIprotein.

In some embodiments, an antibody disclosed herein binds to a CD163protein on a macrophage and is internalized by the macrophage.

In some embodiments, an antibody disclosed herein is not cytotoxic to amacrophage to which it is bound.

In some embodiments, an antibody disclosed herein promotes CD4⁺ T cellactivity or proliferation. In some embodiments, the antibody promotesexpression of CD69, ICOS, OX40, PD1, LAG3, or CTLA4 by CD4⁺ T cells.

In some embodiments, an antibody disclosed herein promotes CD8⁺ T cellactivity or proliferation. In some embodiments, the antibody promotesexpression of ICOS, OX40, PD1, LAG3, or CTLA4 by CD8⁺ T cells.

In some embodiments, an antibody disclosed herein promotes tumor cellkilling in a tumor microenvironment by promoting CD8⁺ T cell activity orproliferation. In some embodiments, the antibody promotes cytotoxiclymphocyte-mediated killing of cancer cells. In some embodiments, theantibody promotes NK cell-mediated tumor cell killing.

In some embodiments, an antibody disclosed herein promotes expression ofIL-2 by T cells. In some embodiments, the binding of antibodies of thepresent disclosure to CD163 protein increases CD4⁺ T cells, CD196⁻ Tcells, CXCR3⁺ T cells, CCR4⁻ T cells, or any combination thereof. Insome embodiments, the antibody increases CD4⁺ CD196⁻ CXCR3⁺ CCR4⁻ Tcells.

In some embodiments, an antibody disclosed herein has a constant domainthat binds to an Fc receptor expressed on a macrophage. In someembodiments, the antibody specifically binds huCD163 and has a constantdomain that binds to an Fc receptor. In some embodiments, the antibodyhas a constant domain that binds to an Fc receptor expressed on CD163⁺immunosuppressive myeloid cells such as CD16 (FcγRIIIa) or CD64 (FcγRI).In some embodiments, the huCD163 and Fc receptor are expressed on thesame cell. In some embodiments, the huCD163 and Fc receptor areexpressed on different cells. In some embodiments, the antibody variabledomain specifically binds huCD163 and the antibody constant domainbinding to an Fc receptor simultaneously.

Disclosed herein, in certain embodiments, are antibodies thatspecifically bind to a CD163 protein expressed on human M2 and M2-likemacrophages, wherein said binding results in at least one of thefollowing effects:

-   -   (a) reduced expression of at least one marker by the human        macrophage, wherein the at least one marker is CD16, CD64, TLR2,        or Siglec-15;    -   (b) internalization of the antibody by the human macrophage;    -   (c) activation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof;    -   (d) proliferation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof; and    -   (e) promotion of tumor cell killing in a tumor microenvironment.

Disclosed herein, in certain embodiments, are antibodies thatspecifically bind to a CD163 protein expressed on human M2 and M2-likemacrophages, wherein said binding results in at least two of thefollowing effects:

-   -   (a) reduced expression of at least one marker by the human        macrophage, wherein the at least one marker is CD16, CD64, TLR2,        or Siglec-15;    -   (b) internalization of the antibody by the human macrophage;    -   (c) activation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof;    -   (d) proliferation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof; and    -   (e) promotion of tumor cell killing in a tumor microenvironment.

Disclosed herein, in certain embodiments, are antibodies thatspecifically bind to a CD163 protein expressed on human M2 and M2-likemacrophages, wherein said binding results in at least three of thefollowing effects:

-   -   (a) reduced expression of at least one marker by the human        macrophage, wherein the at least one marker is CD16, CD64, TLR2,        or Siglec-15;    -   (b) internalization of the antibody by the human macrophage;    -   (c) activation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof;    -   (d) proliferation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof; and    -   (e) promotion of tumor cell killing in a tumor microenvironment.

Disclosed herein, in certain embodiments, are antibodies thatspecifically bind to a CD163 protein expressed on human M2 and M2-likemacrophages, wherein said binding results in at least four of thefollowing effects:

-   -   (a) reduced expression of at least one marker by the human        macrophage, wherein the at least one marker is CD16, CD64, TLR2,        or Siglec-15;    -   (b) internalization of the antibody by the human macrophage;    -   (c) activation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof;    -   (d) proliferation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof; and    -   (e) promotion of tumor cell killing in a tumor microenvironment.

Disclosed herein, in certain embodiments, are antibodies thatspecifically bind to a CD163 protein expressed on human M2 and M2-likemacrophages, wherein said binding results in at least five of thefollowing effects:

-   -   (a) reduced expression of at least one marker by the human        macrophage, wherein the at least one marker is CD16, CD64, TLR2,        or Siglec-15;    -   (b) internalization of the antibody by the human macrophage;    -   (c) activation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof;    -   (d) proliferation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof; and    -   (e) promotion of tumor cell killing in a tumor microenvironment.

In some embodiments, an antibody disclosed herein selectively binds tohuman CD163⁺ immunosuppressive myeloid cells in a tumor-associatedmacrophage (TAM) population, in which the antibody specifically binds toa CD163 protein expressed on the M2 macrophages and reduces animmunosuppressive activity of the TAM population.

In some embodiments, an antibody disclosed herein selectively binds tohuman CD163⁺ immunosuppressive myeloid cells in a tumormicroenvironment, in which the antibody specifically binds to a CD163protein expressed on the M2 macrophages and reduces M2macrophage-mediated suppression. In some embodiments, an antibodydisclosed herein is human, humanized, or chimeric. In some embodiments,an antibody disclosed herein is an antigen-binding fragments thereofthat bind as described.

In some embodiments the antibodies of the present disclosure are intactimmunoglobulin molecules, such as, for example, a human antibody, aswell as those portions of a humanized Ig molecule that contain theantigen-binding site (i.e., paratope) or a single heavy chain and asingle light chain, including those portions known in the art as Fab,Fab′, F(ab)′, F(ab′)2, Fd, scFv, a variable heavy domain, a variablelight domain, a variable NAR domain, bi-specific scFv, a bi-specificFab2, a tri-specific Fab3 a single chain binding polypeptide, a dAbfragment, a diabody, and others also referred to as antigen-bindingfragments. When constructing an immunoglobulin molecule or fragmentsthereof, variable regions or portions thereof are, in some embodiments,fused to, connected to, or otherwise joined to one or more constantregions or portions thereof to produce any of the antibodies orfragments thereof described herein. Thus, in some embodiments, theantigen-binding fragment of any one of the antibodies described above isa Fab, a Fab′, a Fd, a F(ab′)2, a Fv, a scFv, a single chain bindingpolypeptide (e.g., a scFv with Fc portion) or any other functionalfragment thereof as described herein.

In some embodiments, antibodies of the present disclosure are of anyimmunoglobulin class, and, therefore, in some embodiments, have a gamma,mu, alpha, delta, or epsilon heavy chain. In some embodiments, the gammachain is gamma 1, gamma 2, gamma 3, or gamma 4. In some embodiments, thealpha chain is alpha 1 or alpha 2.

In some embodiments, an antibody of the present disclosure is an IgGimmunoglobulin. In some embodiments, antibodies of the presentdisclosure are of any IgG subclass. In some embodiments the antibody isIgG1.

In some embodiments, antibodies of the present disclosure comprise avariable light chain that is either kappa or lambda. In someembodiments, the lambda chain is of any of subtype, including, e.g.,lambda 1, lambda 2, lambda 3, and lambda 4. In some embodiments, thelight chain is kappa.

In some embodiments, antibodies disclosed herein comprise a humanvariable framework region and a human constant region. In someembodiments the antibodies comprise a human light chain variableframework region and a human light chain constant region. In someembodiments the antibodies comprise a human heavy chain variableframework region and a human heavy chain constant region. In someembodiments the antibodies comprise a human light chain variableframework region, a human light chain constant region, a human heavychain variable framework region, and a human heavy chain constantregion.

In some embodiments, the human heavy chain constant region is IgG1 orIgG4 or a fragment thereof. In some embodiments, the heavy chainconstant region is human IgG1. One example of an antibody having an IgG1is AB101. AB101 comprises a light chain comprising SEQ ID NO: 9 and aheavy chain comprising SEQ ID NO: 10, as described in Example 1 below.

In some embodiments, the heavy chain constant region is human IgG1 thathas reduced ADCC function (i.e., a Fc-null antibody). An exemplaryFc-null antibody of the present disclosure is AB102. AB102 comprises alight chain comprising SEQ ID NO: 9 and a heavy chain comprising SEQ IDNO: 11, which contains the variable regions of AB101 and in which theheavy chain constant region is a Fc-null form of human IgG1. AB102 isdescribed further in the examples below.

In some embodiments, the heavy chain constant region is a human IgG1modified to enhance ADCC function. An exemplary antibody of the presentdisclosure having enhanced ADCC function is AB103. AB103 comprises alight chain comprising SEQ ID NO: 9 and a heavy chain comprising SEQ IDNO: 12, which contains the variable regions of AB101 and in which theheavy chain constant region is an enhanced ADCC form of human IgG1.

In some embodiments, the heavy chain constant region is a human IgG4. Anexemplary antibody of the present disclosure having an IgG4 is AB104.AB104 comprises a light chain comprising SEQ ID NO: 9 and a heavy chaincomprising SEQ ID NO: 13, which contains the variable regions of AB101and in which the heavy chain constant region is a human IgG4.

In some embodiments, an antibody of the present disclosure comprises ahuman variable framework region and a murine constant region. In someembodiments, an antibody of the present disclosure comprises a humanheavy chain variable framework region and a murine heavy chain constantregion. In some embodiments, an antibody of the present disclosurecomprises a human light chain variable framework region, a murine lightchain constant region, a human heavy chain variable framework region,and a murine heavy chain constant region.

In some embodiments, the heavy chain constant region is murine IgG2A.One example of an antibody having a murine IgG2A is AB211. AB211comprises a light chain comprising SEQ ID NO: 14 and a heavy chaincomprising SEQ ID NO: 15, which contains the human variable regions ofAB101 and in which the heavy chain constant region is a Fc-null form ofmurine IgG1 and the light chain constant region is a murine kappa. AB211is described further in the examples below.

In some embodiments, the heavy chain constant region is murine IgG2A.One example of an antibody having the heavy chain of a murine IgG2A isAB212. AB212 comprises a light chain comprising SEQ ID NO: 14 and aheavy chain comprising SEQ ID NO: 16, which contains the human variableregions of AB101 and in which the heavy chain constant region is amurine IgG2a and the light chain constant region is a murine kappa.AB212 is described further in the examples below.

Binding of an antibody or antigen-binding fragment to a CD163 proteinexpressed on M2 macrophages are partially (e.g., 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or any number therein) orcompletely modulate a biological function of such M2 macrophages in someembodiments. The activity of an antibody or antigen-binding fragment,for example, are determined using an in vitro assay and/or in vivo usingart-recognized assays such as those described herein or otherwise knownin the art.

In some embodiments, antibodies of the present disclosure are furthermodified to alter the specific properties of the antibody whileretaining the desired functionality, if needed. For example, in oneembodiment, an antibody of the present disclosure is modified to alter apharmacokinetic property of the antibody, including, but not limited to,in vivo stability, solubility, bioavailability, or half-life.

In some embodiments, an antibody described herein has a dissociationconstant (Kd) of about 1 to about 10 pM, from about 10 to about 20 pM,from about 1 to about 29 pM, from about 30 to about 40 pM, from about 10to about 100 pM, or from about 20 to about 500 pM.

In some embodiments, an antibody described herein has a dissociationconstant (Kd) of less than about 500 pM, less than about 400 pM, lessthan about 300 pM, less than about 200 pM, less than about 100 pM, lessthan about 75 pM, less than about 50 pM, less than about 30 pM, lessthan about 25 pM, less than about 20 pM, less than about 18 pM, lessthan about 15 pM, less than about 10 pM, less than about 7.5 pM, lessthan about 5 pM, less than about 2.5 pM, or less than about 1 pM.

In some embodiments, an antibody described herein has an affinity for ahuCD163 protein or peptide of from about 10⁻⁹ to about 10⁻¹⁴, from about10⁻¹⁰ to about 10⁻¹⁴, from about 10⁻¹¹ to about 10⁻¹⁴, from about 10⁻¹²to about 10⁻¹⁴, from about 10⁻¹³ to about 10⁻¹⁴, from about 10⁻¹⁰ toabout 10⁻¹¹, from about 10⁻¹¹ to about 10⁻¹², from about 10⁻¹² to about10⁻¹³, or 10⁻¹³ to about 10⁻¹⁴.

In some embodiments, an antibody described herein has more than onebinding site. In some embodiments, the binding sites are identical toone another. In some embodiments, the binding sites are different fromone another. A naturally occurring human immunoglobulin typically hastwo identical binding sites, while engineered antibodies, for example,have two or more different binding sites.

In some embodiments, an antibody of the present disclosure is bispecificor multi-specific. Bispecific antibodies are antibodies that havebinding specificities for at least two different epitopes. Exemplarybispecific antibodies, in some embodiments, bind to two differentepitopes of a single antigen. Other such antibodies, in someembodiments, combine a first antigen binding site with a binding sitefor a second antigen. In some embodiments, the bispecific antibodiesbind at least two different epitopes and have constant domains that bindto Fc receptors. In some embodiments, the binding of one or moreepitopes of the bispecific antibodies is simultaneous with binding ofthe constant domains of the bispecific antibodies to Fc receptors.

In some embodiments, an antibody of the present disclosure has two ormore valencies, which are also referred to as multivalent. In someembodiments, an antibody of the present disclosure is trispecific. Insome embodiments, the multivalent antibody is internalized (and/orcatabolized) faster than a bivalent antibody by a cell expressing anantigen to which the antibodies bind. In some embodiments, theantibodies of the present disclosure are multivalent antibodies withthree or more antigen binding sites (e.g., tetravalent antibodies). Insome embodiments, the multivalent antibodies of the present disclosureare produced by recombinant expression of nucleic acid encoding thepolypeptide chains of the antibody. In some embodiments, the multivalentantibody comprises a dimerization domain and three or more antigenbinding sites. In some embodiments, the dimerization domain comprises(or consists of) an Fc region or a hinge region. In this scenario, theantibody will comprise an Fc region and three or more antigen bindingsites amino-terminal to the Fc region. In some embodiments, themultivalent antibody herein comprises about three to about eight, butpreferably four, antigen binding sites. The multivalent antibodycomprises at least one polypeptide chain (and preferably two polypeptidechains), wherein the polypeptide chain(s) comprise two or more variableregions. For instance, the polypeptide chain(s) comprisesVD1-(X1)_(n)-VD2-(X2)_(n)-Fc, wherein VD1 is a first variable region,VD2 is a second variable region, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. In some embodiments, the polypeptide chain(s) each independentlycomprise: V_(H)-C_(H)1-flexible linker-V_(H)-C_(H)1-Fc region chain; orV_(H)-C_(H)1-V_(H)-C_(H)1-Fc region chain. In some embodiments, themultivalent antibody herein further comprises at least two (andpreferably four) light chain variable region polypeptides. In someembodiments, the multivalent antibody herein comprises from about two toabout eight light chain variable region polypeptides. In someembodiments, the light chain variable region polypeptides describedherein comprise a light chain variable region. In some embodiments, thelight chain variable region polypeptides described herein furthercomprise a C_(L) domain.

In some embodiments, an antibody of the present disclosure isconstructed to fold into multivalent forms, which, in some embodiments,improves binding affinity, specificity, and/or increased half-life inblood. Multivalent forms of antibodies are prepared, for example, bytechniques known in the art.

In some embodiments, an antibody of the present disclosure is an SMIP orbinding domain immunoglobulin fusion protein specific for the targetprotein. These constructs are single-chain polypeptides comprisingantigen-binding domains fused to immunoglobulin domains necessary tocarry out antibody effector functions.

In some embodiments, an antibody of the present disclosure comprises asingle chain binding polypeptide having a heavy chain variable region,and/or a light chain variable region which binds an epitope disclosedherein and has, optionally, an immunoglobulin Fc region. Such a moleculeis a single chain variable fragment (scFv) optionally having effectorfunction or increased half-life through the presence of theimmunoglobulin Fc region.

Anti-CD163 Antibodies

Provided herein, in certain embodiments, are antibodies thatspecifically bind to CD163 proteins. In some embodiments, CD163-bindingantibodies comprise at least one heavy chain and at least one lightchain. In some embodiments, CD163-binding antibodies comprise at leastone heavy chain comprising a heavy chain variable domain (VH) and atleast one light chain comprising a light chain variable domain (VL).Each VH and VL comprises three complementarity determining regions(CDR). The amino acid sequences of the VH and VL and the CDRs determinethe antigen binding specificity and antigen binding strength of theantibody. The VH and VL domains are summarized in Table 1. The lightchains and heavy chains are summarized in Table 2. The amino acidsequences of the CDRs are summarized in Table 3.

In some embodiments, an antibody disclosed herein is a monoclonalantibody. In some embodiments, an antibody disclosed herein is anantigen binding fragment. In some embodiments, an antibody disclosedherein is selected from a whole immunoglobulin, an scFv, a Fab, aF(ab′)2, or a disulfide linked Fv. In some embodiments, an antibodydisclosed herein is an IgG or an IgM. In some embodiments, an antibodydisclosed herein is humanized. In some embodiments, an antibodydisclosed herein is chimeric.

Anti-CD163 Antibody Variable Domains

TABLE 1 Anti-CD163 Variable Domain Sequences.Table 1: Anti-CD163 Variable Domain Sequences SEQ ID SEQUENCE NO:Light Chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPK 7Variable domain LLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPRGTFGQGTKVEIKR Heavy ChainEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGK 8 Variable domainGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARENVRPYYDFWSGYYSEYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF

Disclosed herein, in certain embodiments, are antibodies comprising alight chain variable domain (VL) having an amino acid sequence at leastabout 70% identical to an amino acid sequence set forth as SEQ ID NO: 7.In some embodiments the VL has an amino acid sequence at least about75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequenceset forth as SEQ ID NO: 7. In some embodiments, the VL has an amino acidsequence 100% identical to an amino acid sequence set forth as SEQ IDNO: 7.

Disclosed herein, in certain embodiments, are antibodies comprising aheavy chain variable domain (VH) having an amino acid sequence at leastabout 70% identical to an amino acid sequence set forth as SEQ ID NO: 8.In some embodiments the VH has an amino acid sequence at least about75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequenceset forth as SEQ ID NO: 8. In some embodiments, the VH has an amino acidsequence 100% identical to an amino acid sequence set forth as SEQ IDNO: 8.

Disclosed herein, in certain embodiments, are antibodies comprising alight chain variable domain (VL) having an amino acid sequence at leastabout 70% identical to an amino acid sequence set forth as SEQ ID NO: 7and a heavy chain variable domain (VH) having an amino acid sequence atleast about 70% identical to an amino acid sequence set forth as SEQ IDNO: 8. In some embodiments the VL has an amino acid sequence at leastabout 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acidsequence set forth as SEQ ID NO: 7 and the VH has an amino acid sequenceat least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to anamino acid sequence set forth as SEQ ID NO: 8. In some embodiments, theVL has an amino acid sequence 100% identical to an amino acid sequenceset forth as SEQ ID NO: 7 and the VH has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 8.

Anti-CD163 Antibody Light Chain and Heavy Chain

TABLE 2 Anti-CD163 Light Chain and Heavy Chain SequencesTable 2: Anti-CD163 Light Chain and Heavy Chain Sequences SEQ IDSEQUENCE NO: AB101, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP  9AB102, KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ AB103, orQSYSTPRGTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV AB104 LightVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS Chain (hIgG1)LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AB101 HeavyEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPG 10 Chain (hIgG1)KGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARENVRPYYDFWSGYYSEYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AB102 HeavyEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPG 11 Chain (hIgG1KGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMN ADCC-Null)SLRAEDTAVYYCARENVRPYYDFWSGYYSEYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AB103 HeavyEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPG 12 Chain (hIgG1KGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMN eADCC)SLRAEDTAVYYCARENVRPYYDFWSGYYSEYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AB104 HeavyEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPG 13 Chain (hIgG4)KGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARENVRPYYDFWSGYYSEYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK AB211 orDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP 14 AB212 LightKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ ChainQSYSTPRGTFGQGTKVEIKRTDAAPTVSIFPPSSEQLTSGGASVV (muKappa)CFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC AB211 HeavyEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPG 15 ChainKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMN (muIgG2A)SLRAEDTAVYYCARENVRPYYDFWSGYYSEYYYYGMDVWGQGTTVTVSSKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK AB212 HeavyEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPG 16 ChainKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMN (muIgG2ASLRAEDTAVYYCARENVRPYYDFWSGYYSEYYYYGMDVWG ADCC-Null)QGTTVTVSSKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLEGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKAFACAVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

Disclosed herein, in certain embodiments, are antibodies comprising alight chain having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 9. In some embodimentsthe light chain has an amino acid sequence at least about 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to an amino acid sequence set forth asSEQ ID NO: 9. In some embodiments, the light chain has an amino acidsequence 100% identical to an amino acid sequence set forth as SEQ IDNO: 9.

Disclosed herein, in certain embodiments, are antibodies comprising aheavy chain having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 10. In someembodiments the heavy chain has an amino acid sequence at least about75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequenceset forth as SEQ ID NO: 10. In some embodiments, the heavy chain has anamino acid sequence 100% identical to an amino acid sequence set forthas SEQ ID NO: 10.

Disclosed herein, in certain embodiments, are antibodies comprising aheavy chain having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 11. In someembodiments the heavy chain has an amino acid sequence at least about75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequenceset forth as SEQ ID NO: 11. In some embodiments, the heavy chain has anamino acid sequence 100% identical to an amino acid sequence set forthas SEQ ID NO: 11.

Disclosed herein, in certain embodiments, are antibodies comprising aheavy chain having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 12. In someembodiments the heavy chain has an amino acid sequence at least about75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequenceset forth as SEQ ID NO: 12. In some embodiments, the heavy chain has anamino acid sequence 100% identical to an amino acid sequence set forthas SEQ ID NO: 12.

Disclosed herein, in certain embodiments, are antibodies comprising aheavy chain having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 13. In someembodiments the heavy chain has an amino acid sequence at least about75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequenceset forth as SEQ ID NO: 13. In some embodiments, the heavy chain has anamino acid sequence 100% identical to an amino acid sequence set forthas SEQ ID NO: 13.

Disclosed herein, in certain embodiments, are antibodies comprising alight chain having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 9 and a heavy chainhaving an amino acid sequence at least about 70% identical to an aminoacid sequence set forth as SEQ ID NO: 10. In some embodiments the lightchain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence set forth as SEQ ID NO:9 and the heavy chain has an amino acid sequence at least about 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence setforth as SEQ ID NO: 10. In some embodiments, the light chain has anamino acid sequence 100% identical to an amino acid sequence set forthas SEQ ID NO: 9 and the heavy chain has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 10.

Disclosed herein, in certain embodiments, are antibodies comprising alight chain having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 9 and a heavy chainhaving an amino acid sequence at least about 70% identical to an aminoacid sequence set forth as SEQ ID NO: 11. In some embodiments the lightchain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence set forth as SEQ ID NO:9 and the heavy chain has an amino acid sequence at least about 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence setforth as SEQ ID NO: 11. In some embodiments, the light chain has anamino acid sequence 100% identical to an amino acid sequence set forthas SEQ ID NO: 9 and the heavy chain has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 11.

Disclosed herein, in certain embodiments, are antibodies comprising alight chain having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 9 and a heavy chainhaving an amino acid sequence at least about 70% identical to an aminoacid sequence set forth as SEQ ID NO: 12. In some embodiments the lightchain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence set forth as SEQ ID NO:9 and the heavy chain has an amino acid sequence at least about 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence setforth as SEQ ID NO: 12. In some embodiments, the light chain has anamino acid sequence 100% identical to an amino acid sequence set forthas SEQ ID NO: 9 and the heavy chain has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 12.

Disclosed herein, in certain embodiments, are antibodies comprising alight chain having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 9 and a heavy chainhaving an amino acid sequence at least about 70% identical to an aminoacid sequence set forth as SEQ ID NO: 13. In some embodiments the lightchain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence set forth as SEQ ID NO:9 and the heavy chain has an amino acid sequence at least about 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence setforth as SEQ ID NO: 13. In some embodiments, the light chain has anamino acid sequence 100% identical to an amino acid sequence set forthas SEQ ID NO: 9 and the heavy chain has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 13.

Disclosed herein, in certain embodiments, are antibodies comprising alight chain having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 14. In someembodiments the light chain has an amino acid sequence at least about75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequenceset forth as SEQ ID NO: 14. In some embodiments, the light chain has anamino acid sequence 100% identical to an amino acid sequence set forthas SEQ ID NO: 14.

Disclosed herein, in certain embodiments, are antibodies comprising aheavy chain having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 15. In someembodiments the heavy chain has an amino acid sequence at least about75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequenceset forth as SEQ ID NO: 15. In some embodiments, the heavy chain has anamino acid sequence 100% identical to an amino acid sequence set forthas SEQ ID NO: 15.

Disclosed herein, in certain embodiments, are antibodies comprising aheavy chain having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 16. In someembodiments the heavy chain has an amino acid sequence at least about75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequenceset forth as SEQ ID NO: 16. In some embodiments, the heavy chain has anamino acid sequence 100% identical to an amino acid sequence set forthas SEQ ID NO: 16.

Disclosed herein, in certain embodiments, are antibodies comprising alight chain having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 14 and a heavy chainhaving an amino acid sequence at least about 70% identical to an aminoacid sequence set forth as SEQ ID NO: 15. In some embodiments the lightchain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence set forth as SEQ ID NO:14 and the heavy chain has an amino acid sequence at least about 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence setforth as SEQ ID NO: 15. In some embodiments, the light chain has anamino acid sequence 100% identical to an amino acid sequence set forthas SEQ ID NO: 14 and the heavy chain has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 15.

Disclosed herein, in certain embodiments, are antibodies comprising alight chain having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 14 and a heavy chainhaving an amino acid sequence at least about 70% identical to an aminoacid sequence set forth as SEQ ID NO: 16. In some embodiments the lightchain has an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence set forth as SEQ ID NO:14 and the heavy chain has an amino acid sequence at least about 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence setforth as SEQ ID NO: 16. In some embodiments, the light chain has anamino acid sequence 100% identical to an amino acid sequence set forthas SEQ ID NO: 14 and the heavy chain has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 16.

Exemplary Anti-CD163 Complementarity Determining Regions

TABLE 3 anti-CD163 CDR Sequences Table 3: Anti-CD163 CDR SequencesSEQ ID SEQUENCE NO: Light Chain RASQSISSYLN 1 CDR1 Light Chain AASSLQS 2CDR2 Light Chain QQSYSTPRGT 3 CDR3 Heavy Chain SYAMH 4 CDR1 Heavy ChainVISYDGSNKYYADSVKG 5 CDR2 Heavy Chain ENVRPYYDFWSGYYSEYYYYGMDV 6 CDR3

Disclosed herein, in certain embodiments, are antibodies comprising atleast one of a light chain CDR1 having an amino acid sequence at leastabout 70% identical to an amino acid sequence set forth as SEQ ID NO: 1,a light chain CDR2 having an amino acid sequence at least about 70%identical to an amino acid sequence set forth as SEQ ID NO: 2, and alight chain CDR3 having an amino acid sequence at least about 70%identical to an amino acid sequence set forth as SEQ ID NO: 3. In someembodiments, antibodies binding to CD163 comprise at least one of alight chain CDR1 having an amino acid sequence at least about 75%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forthas SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence atleast about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an aminoacid sequence set forth as SEQ ID NO: 2, and a light chain CDR3 havingan amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence set forth as SEQ ID NO: 3. In someembodiments, antibodies binding to CD163 comprise at least one of alight chain CDR1 having an amino acid sequence 100% identical to anamino acid sequence set forth as SEQ ID NO: 1, a light chain CDR2 havingan amino acid sequence 100% identical to an amino acid sequence setforth as SEQ ID NO: 2, and a light chain CDR3 having an amino acidsequence 100% identical to an amino acid sequence set forth as SEQ IDNO: 3.

Disclosed herein, in certain embodiments, are antibodies comprising atleast one of a heavy chain CDR1 having an amino acid sequence at leastabout 70% identical to an amino acid sequence set forth as SEQ ID NO: 4,a heavy chain CDR2 having an amino acid sequence at least about 70%identical to an amino acid sequence set forth as SEQ ID NO: 5, a heavychain CDR3 having an amino acid sequence at least about 70% identical toan amino acid sequence set forth as SEQ ID NO: 6. In some embodiments,antibodies binding to CD163 comprise at least one of a heavy chain CDR1having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence set forth as SEQ ID NO:4, a heavy chain CDR2 having an amino acid sequence at least about 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence setforth as SEQ ID NO: 5, a heavy chain CDR3 having an amino acid sequenceat least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to anamino acid sequence set forth as SEQ ID NO: 6. In some embodiments,antibodies binding to CD163 comprise at least one of a heavy chain CDR1having an amino acid sequence 100% identical to an amino acid sequenceset forth as SEQ ID NO: 4, a heavy chain CDR2 having an amino acidsequence 100% identical to an amino acid sequence set forth as SEQ IDNO: 5, a heavy chain CDR3 having an amino acid sequence 100% identicalto an amino acid sequence set forth as SEQ ID NO: 6.

Disclosed herein, in certain embodiments, are antibodies comprising atleast one of a light chain CDR1 having an amino acid sequence at leastabout 70% identical to an amino acid sequence set forth as SEQ ID NO: 1,a light chain CDR2 having an amino acid sequence at least about 70%identical to an amino acid sequence set forth as SEQ ID NO: 2, a lightchain CDR3 having an amino acid sequence at least about 70% identical toan amino acid sequence set forth as SEQ ID NO: 3, a heavy chain CDR1having an amino acid sequence at least about 70% identical to an aminoacid sequence set forth as SEQ ID NO: 4, a heavy chain CDR2 having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 5, and a heavy chain CDR3 having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 6. In some embodiments, antibodiesbinding to CD163 comprise at least one of a light chain CDR1 having anamino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence set forth as SEQ ID NO: 1, a lightchain CDR2 having an amino acid sequence at least about 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to an amino acid sequence set forth asSEQ ID NO: 2, a light chain CDR3 having an amino acid sequence at leastabout 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acidsequence set forth as SEQ ID NO: 3, a heavy chain CDR1 having an aminoacid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence set forth as SEQ ID NO: 4, a heavychain CDR2 having an amino acid sequence at least about 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to an amino acid sequence set forth asSEQ ID NO: 5, and a heavy chain CDR3 having an amino acid sequence atleast about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an aminoacid sequence set forth as SEQ ID NO: 6. In some embodiments, antibodiesbinding to CD163 comprise at least one of a light chain CDR1 having anamino acid sequence 100% identical to an amino acid sequence set forthas SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 2, a lightchain CDR3 having an amino acid sequence 100% identical to an amino acidsequence set forth as SEQ ID NO: 3, a heavy chain CDR1 having an aminoacid sequence 100% identical to an amino acid sequence set forth as SEQID NO: 4, a heavy chain CDR2 having an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 5, and aheavy chain CDR3 having an amino acid sequence 100% identical to anamino acid sequence set forth as SEQ ID NO: 6.

Binding Affinity and Immunoreactivity

Binding affinity and/or avidity of antibodies or antigen-bindingfragments thereof are improved by modifying framework regions. Anysuitable methods for modifications of framework regions are known in theart and are contemplated herein. Selection of one or more relevantframework amino acid positions to alter depends on a variety ofcriteria. One criterion for selecting relevant framework amino acids tochange is, for example, the relative differences in amino acid frameworkresidues between the donor and acceptor molecules. Selection of relevantframework positions to alter using this approach has the advantage ofavoiding any subjective bias in residue determination or any bias in CDRbinding affinity contribution by the residue.

Binding interactions are manifested as an intermolecular contact withone or more amino acid residues of one or more CDRs in some embodiments.Antigen-binding involves, for example, a CDR or a CDR pair or, in somecases, interactions of up to all six CDRs of the V_(H) and V_(L) chains.

Binding affinity and avidity of antibodies or antigen-binding fragmentscan be measured by surface plasmon resonance (SPR) measurements,AlphaLisa assays or flow cytometry of the equilibrium dissociationconstant (K_(D)).

Disclosed herein are antibodies that specifically bind to human CD163with a K_(D) from 0.1 nM to 1000 nM. In some embodiments, the antibodiesspecifically bind to human CD163 with a K_(D) from about 0.1 to about500 nM, from about 0.1 to about 100 nM, from about 0.1 to about 50 nM,from about 0.1 to about 20 nM, from about 0.1 to about 10 nM, from about0.1 to about 5 nM, from about 0.1 to about 2 nM, from about 0.1 to about1 nM, from about 0.1 to about 0.5 nM, from about 0.5 to about 1000 nM,from about 0.5 to about 500 nM, from about 0.5 to about 100 nM, fromabout 0.5 to about 50 nM, from about 0.5 to about 20 nM, from about 0.5to about 10 nM, from about 0.5 to about 5 nM, from about 0.5 to about 2nM, from about 0.5 to about 1 nM, from about 1 to about 1000 nM, fromabout 1 to about 500 nM, from about 1 to about 100 nM, from about 1 toabout 50 nM, from about 1 to about 20 nM, from about 1 to about 10 nM,from about 1 to about 5 nM, from about 1 to about 2 nM, from about 2 toabout 1000 nM, from about 2 to about 500 nM, from about 2 to about 100nM, from about 2 to about 50 nM, from about 2 to about 20 nM, from about2 to about 10 nM, from about 2 to about 5 nM, from about 5 to about 1000nM, from about 5 to about 500 nM, from about 5 to about 100 nM, fromabout 5 to about 50 nM, from about 5 to about 20 nM, from about 5 toabout 10 nM, from about 10 to about 1000 nM, from about 10 to about 500nM, from about 10 to about 100 nM, from about 10 to about 50 nM, fromabout 10 to about 20 nM, from about 20 to about 1000 nM, from about 20to about 500 nM, from about 20 to about 100 nM, from about 20 to about50 nM, from about 50 to about 1000 nM, from about 50 to about 500 nM,from about 50 to about 100 nM, from about 100 to about 500 nM, fromabout 100 to about 1000 nM, from about 500 to about 1000 nM. In someembodiments, the antibodies specifically bind to human CD163 with aK_(D) of 1.8 nM, 12 nM, 45 nM or 89 nM.

The antibodies disclosed herein binds to the myeloid scavenger receptorCD163, which is highly expressed on tumor associated macrophages (TAMs)and has been detected on various tumor cells from different origins. Theexpression of CD163 in tumor tissue is associated with poor prognosis.The binding affinity between the antibodies disclosed herein and IL-10polarized M2c macrophages are measured by flow cytometry assays.

Disclosed herein are antibodies that specifically binds to M2cmacrophages with a K_(D) from 0.1 nM to 1000 nM. In some embodiments,the antibodies specifically bind to M2c macrophages with a K_(D) fromabout 0.1 to about 500 nM, from about 0.1 to about 100 nM, from about0.1 to about 50 nM, from about 0.1 to about 20 nM, from about 0.1 toabout 10 nM, from about 0.1 to about 5 nM, from about 0.1 to about 2 nM,from about 0.1 to about 1 nM, from about 0.1 to about 0.5 nM, from about0.5 to about 1000 nM, from about 0.5 to about 500 nM, from about 0.5 toabout 100 nM, from about 0.5 to about 50 nM, from about 0.5 to about 20nM, from about 0.5 to about 10 nM, from about 0.5 to about 5 nM, fromabout 0.5 to about 2 nM, from about 0.5 to about 1 nM, from about 1 toabout 1000 nM, from about 1 to about 500 nM, from about 1 to about 100nM, from about 1 to about 50 nM, from about 1 to about 20 nM, from about1 to about 10 nM, from about 1 to about 5 nM, from about 1 to about 2nM, from about 2 to about 1000 nM, from about 2 to about 500 nM, fromabout 2 to about 100 nM, from about 2 to about 50 nM, from about 2 toabout 20 nM, from about 2 to about 10 nM, from about 2 to about 5 nM,from about 5 to about 1000 nM, from about 5 to about 500 nM, from about5 to about 100 nM, from about 5 to about 50 nM, from about 5 to about 20nM, from about 5 to about 10 nM, from about 10 to about 1000 nM, fromabout 10 to about 500 nM, from about 10 to about 100 nM, from about 10to about 50 nM, from about 10 to about 20 nM, from about 20 to about1000 nM, from about 20 to about 500 nM, from about 20 to about 100 nM,from about 20 to about 50 nM, from about 50 to about 1000 nM, from about50 to about 500 nM, from about 50 to about 100 nM, from about 100 toabout 500 nM, from about 100 to about 1000 nM, from about 500 to about1000 nM. In some embodiments, the antibodies specifically bind to M2cmacrophages with a K_(D) of 7.7 nM.

Binding Epitopes

Antibody epitopes may be a linear peptide sequence (i.e., “continuous”)or may be composed of noncontiguous amino acid sequences (i.e.,“conformational” or “discontinuous”). In some embodiments, an antibodyrecognizes one or more amino acid sequences; therefore, an epitopedefines more than one distinct amino acid sequence. Epitopes recognizedby antibodies are determined, for example, by peptide mapping andsequence analysis techniques well known to one of skill in the art.Binding interactions are manifested as intermolecular contacts with oneor more amino acid residues of a CDR.

Human CD163 protein is a protein that in humans is encoded by the CD163gene. The amino acid sequence of human CD163 is:

(SEQ ID NO: 17) MSKLRMVLLEDSGSADFRRHFVNLSPFTITVVLLLSACFVTSSLGGTDKELRLVDGENKCSGRVEVKVQEEWGTVCNNGWSMEAVSVICNQLGCPTAIKAPGWANSSAGSGRIWMDHVSCRGNESALWDCKHDGWGKHSNCTHQQDAGVTCSDGSNLEMRLTRGGNMCSGRIEIKFQGRWGTVCDDNFNIDHASVICRQLECGSAVSFSGSSNFGEGSGPIWFDDLICNGNESALWNCKHQGWGKHNCDHAEDAGVICSKGADLSLRLVDGVTECSGRLEVRFQGEWGTICDDGWDSYDAAVACKQLGCPTAVTAIGRVNASKGFGHIWLDSVSCQGHEPAIWQCKHHEWGKHYCNHNEDAGVTCSDGSDLELRLRGGGSRCAGTVEVEIQRLLGKVCDRGWGLKEADVVCRQLGCGSALKTSYQVYSKIQATNTWLFLSSCNGNETSLWDCKNWQWGGLTCDHYEEAKITCSAHREPRLVGGDIPCSGRVEVKHGDTWGSICDSDFSLEAASVLCRELQCGTVVSILGGAHFGEGNGQIWAEEFQCEGHESHLSLCPVAPRPEGTCSHSRDVGVVCSRYTEIRLVNGKTPCEGRVELKTLGAWGSLCNSHWDIEDAHVLCQQLKCGVALSTPGGARFGKGNGQIWRHMFHCTGTEQHMGDCPVTALGASLCPSEQVASVICSGNQSQTLSSCNSSSLGPTRPTIPEESAVACIESGQLRLVNGGGRCAGRVEIYHEGSWGTICDDSWDLSDAHVVCRQLGCGEAINATGSAHFGEGTGPIWLDEMKCNGKESRIWQCHSHGWGQQNCRHKEDAGVICSEFMSLRLTSEASREACAGRLEVFYNGAWGTVGKSSMSETTVGVVCRQLGCADKGKINPASLDKAMSIPMWVDNVQCPKGPDTLWQCPSSPWEKRLASPSEETWITCDNKIRLQEGPTSCSGRVEIWHGGSWGTVCDDSWDLDDAQVVCQQLGCGPALKAFKEAEFGQGTGPIWLNEVKCKGNESSLWDCPARRWGHSECGHKEDAAVNCTDISVQKTPQKATTGRSSRQSSFIAVGILGVVLLAIFVALFFLTKKRRQRQRLAVSSRGENLVHQIQYREMNSCLNADDLDLMNSSGGHSEPH.

Disclosed herein are antibodies that specifically bind to an epitope inhuman CD163. In some embodiments, an antibody disclosed herein binds toan epitope comprising noncontiguous amino acid sequences. In someembodiments, the antibody binds to an epitope of human CD163 comprisingthe amino acid sequence IGRVNASKGFGHIWLDSVSCQGHEPAI (SEQ ID NO: 18). Insome embodiments, the antibody binds to an epitope of human CD163comprising the amino acid sequence VVCRQLGCGSA (SEQ ID NO: 19). In someembodiments, the antibody binds to an epitope of human CD163 comprisingthe amino acid sequence WDCKNWQWGGLTCD (SEQ ID NO: 20). In someembodiments, the antibody binds to an epitope of human CD163 comprisingthe amino acid sequences of SEQ ID NOs:18-20.

Also disclosed herein are additional antibodies that specifically bindto the epitope disclosed herein. These additional antibodies, orantigen-binding fragments thereof that specifically bind to the epitopedisclosed herein can be identified using techniques known in the art.For example, a computational approach is used to design epitope-specificantibodies. Nimrod et al., Computational Design of Epitope-SpecificFunctional Antibodies, Cell Reports 25, 2121-2131, Nov. 20, 2018,(incorporated herein by reference). Another approach can be used toidentify antibodies that bind to specific epitopes from a library ofantibodies that bind to the antigen, such as the following: firstincorporate noncanonical amino acids (ncAAs) p-benzoyl-L-phenylalanine(pBpa) and p-azido-L-phenylalanine (pAzF) into the target epitope andthen select the antibodies that cross-link with the ncAA incorporatedepitope after UV irradiation. Because cross-linking only occurs when thedistance between the antibody and the epitope is close enough, thismethod can efficiently select antibodies that specifically bind to thetarget epitope. Chen et al. Epitope-directed antibody selection bysite-specific photocrosslinking, Science Advances, Vol. 6, no. 14,eaaz7825, 1 Apr. 2020 (incorporated herein by reference).

Modifications of Antibodies

Antibodies, or antigen-binding fragments thereof, are modified, in somecases, using techniques known in the art for various purposes such as,for example, by addition of polyethylene glycol (PEG). In someembodiments, PEG modification (PEGylation) leads to one or more ofimproved circulation time, improved solubility, improved resistance toproteolysis, reduced antigenicity and immunogenicity, improvedbioavailability, reduced toxicity, improved stability, and easierformulation.

In some cases when an antigen-binding fragment does not contain an Fcportion, an Fc portion is added to (e.g., recombinantly) the fragment,for example, to increase half-life of the antigen-binding fragment incirculation in blood when administered to a subject. Choice of anappropriate Fc region and methods of to incorporate such fragments areknown in the art. Incorporating an Fc region of an IgG into apolypeptide of interest so as to increase its circulatory half-life, butso as not to lose its biological activity is accomplished, for example,by using conventional techniques known in the art. In some embodiments,Fc portions of antibodies are further modified to increase half-life ofthe antigen-binding fragment in circulation in blood when administeredto a subject. Modifications are, for example, determined usingconventional means in the art.

Additionally, in some embodiments, antibodies and antigen-bindingfragments thereof are produced or expressed so that they do not containfucose on their complex N-glycoside-linked sugar chains. The removal ofthe fucose from the complex N-glycoside-linked sugar chains is known toincrease effector functions of the antibodies and antigen-bindingfragments, including but not limited to, antibody dependentcell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity(CDC). Similarly, antibodies or antigen-binding fragments thereof thatbind an epitope are, in some cases, attached at their C-terminal end toall or part of an immunoglobulin heavy chain derived from any antibodyisotype, e.g., IgG, IgA, IgE, IgD, and IgM, and any of the isotypesubclasses, particularly IgG1, IgG2, IgG3, and IgG4.

Additionally, the antibodies or antigen-binding fragments describedherein are also modified so that they are able to cross the blood-brainbarrier in some embodiments. Such modification of the antibodies orantigen-binding fragments described herein allows for the treatment ofbrain diseases such as glioblastoma multiforme (GBM). Exemplarymodifications to allow proteins such as antibodies or antigen-bindingfragments to cross the blood-brain barrier are described in US Pat.Publ. 2007/0082380.

Glycosylation of immunoglobulins has been shown to have significanteffects on their effector functions, structural stability, and rate ofsecretion from antibody-producing cells. The carbohydrate groupsresponsible for these properties are generally attached to the constant(C) regions of the antibodies. For example, glycosylation of IgG atasparagine 297 in the C_(H)2 domain is required for full capacity of IgGto activate the classical pathway of complement-dependent cytolysis (Taoand Morrison, J Immunol 143:2595 (1989)). Glycosylation of IgM atasparagine 402 in the C_(H)3 domain is necessary for proper assembly andcytolytic activity of the antibody (Muraoka and Shulman, J Immunol142:695 (1989)). Removal of glycosylation sites as positions 162 and 419in the C_(H)1 and C_(H)3 domains of an IgA antibody led to intracellulardegradation and at least 90% inhibition of secretion (Taylor and Wall,Mol Cell Biol 8:4197 (1988)). Additionally, in some embodiments,antibodies and antigen-binding fragments thereof are produced orexpressed so that they do not contain fucose on their complexN-glycoside-linked sugar chains. The removal of the fucose from thecomplex N-glycoside-linked sugar chains is known to increase effectorfunctions of the antibodies and antigen-binding fragments, including butnot limited to, antibody dependent cell-mediated cytotoxicity (ADCC) andcomplement dependent cytotoxicity (CDC). These “defucosylated”antibodies and antigen-binding fragments are produced, in someembodiments, through a variety of systems utilizing molecular cloningtechniques known in the art, including but not limited to, transgenicanimals, transgenic plants, or cell-lines that have been geneticallyengineered so that they no longer contain the enzymes and biochemicalpathways necessary for the inclusion of a fucose in the complexN-glycoside-linked sugar chains (also known as fucosyltransferaseknock-out animals, plants, or cells). Non-limiting examples of cellsthat are engineered to be fucosyltransferase knock-out cells include CHOcells, SP2/0 cells, NS0 cells, and YB2/0 cells.

Glycosylation of immunoglobulins in the variable (V) region has alsobeen observed. Sox and Hood reported that about 20% of human antibodiesare glycosylated in the V region (Proc Natl Acad Sci USA 66:975 (1970)).Glycosylation of the V domain is believed to arise from fortuitousoccurrences of the N-linked glycosylation signal Asn-Xaa-Ser/Thr in theV region sequence and has not been recognized in the art as playing arole in immunoglobulin function.

Glycosylation at a variable domain framework residue, in some cases,alters the binding interaction of the antibody with antigen. The presentdisclosure includes criteria by which a limited number of amino acids inthe framework or CDRs of a humanized immunoglobulin chain are chosen tobe mutated (e.g., by substitution, deletion, or addition of residues) toincrease the affinity of an antibody.

In some embodiments, cysteine residue(s) are removed or introduced inthe Fc region of an antibody or Fc-containing polypeptide, therebyeliminating or increasing interchain disulfide bond formation in thisregion. A homodimeric specific binding agent or antibody generated usingsuch methods, in some embodiments, exhibit improved internalizationcapability and/or increased complement-mediated cell killing andantibody-dependent cellular cytotoxicity (ADCC).

It has been shown that sequences within the CDR cause an antibody tobind to MHC Class II and trigger an unwanted helper T-cell response insome cases. In some embodiments, a conservative substitution allows theantibody to retain binding activity yet reduce its ability to trigger anunwanted T-cell response. In one embodiment, one or more of theN-terminal 20 amino acids of the heavy or light chain is removed.

In some embodiments, antibody molecules are produced with alteredcarbohydrate structure resulting in altered effector activity, includingantibody molecules with absent or reduced fucosylation that exhibitimproved ADCC activity. A variety of ways are known in the art toaccomplish this. For example, ADCC effector activity is mediated bybinding of the antibody molecule to the FcγRIII receptor, which has beenshown to be dependent on the carbohydrate structure of the N-linkedglycosylation at the Asn-297 of the C_(H)2 domain. Non-fucosylatedantibodies bind this receptor with increased affinity and triggerFcγRIII-mediated effector functions more efficiently than native,fucosylated antibodies. Some host cell strains, e.g., Lec13 or rathybridoma YB2/0 cell line naturally produce antibodies with lowerfucosylation levels. An increase in the level of bisected carbohydrate,e.g., through recombinantly producing antibody in cells that overexpressGnTIII enzyme, has also been determined to increase ADCC activity. Insome embodiments, the absence of only one of the two fucose residues aresufficient to increase ADCC activity.

Covalent modifications of an antibody are also included herein. In someembodiments, they are made by chemical synthesis or by enzymatic orchemical cleavage of the antibody, if applicable. In some embodiments,other types of covalent modifications are introduced by reactingtargeted amino acid residues with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues.

Cysteinyl residues most commonly are reacted with alpha-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, alpha-bromo-beta-(5-imidazoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

In some embodiments, histidyl residues are derivatized by reaction withdiethylpyrocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. In some embodiments,para-bromophenacyl bromide also is useful; the reaction, in someembodiments, is performed in 0.1 M sodium cacodylate at pH 6.0.

In some embodiments, lysinyl and amino-terminal residues are reactedwith succinic or other carboxylic acid anhydrides. Derivatization withthese agents has the effect of reversing the charge of the lysinylresidues. Other suitable reagents for derivatizingalpha-amino-containing residues include imidoesters such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitrobenzenesulfonic acid, 0-methylisourea, 2,4-pentanedione, andtransaminase-catalyzed reaction with glyoxylate.

In some embodiments, arginyl residues are modified by reaction with oneor several conventional reagents, such as phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginine residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents, in some embodiments, react with the groupsof lysine as well as the arginine epsilon-amino group.

In some embodiments, the specific modification of tyrosyl residues aremade, with particular interest in introducing spectral labels intotyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidazole andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively, in some embodiments. Tyrosyl residues areiodinated using ¹²⁵I or ¹³¹I to prepare labeled proteins for use inradioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are specifically modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

In some embodiments, glutaminyl and asparaginyl residues are deamidatedto the corresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the alpha-amino groups of lysine, arginine, and histidineside chains, acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the specific binding agent orantibody. These procedures do not require production of the polypeptideor antibody in a host cell that has glycosylation capabilities for N- orO-linked glycosylation. Depending on the coupling mode used, in someembodiments, the sugar(s) are attached to (a) arginine and histidine,(b) free carboxyl groups, (c) free sulfhydryl groups such as those ofcysteine, (d) free hydroxyl groups such as those of serine, threonine,or hydroxyproline, (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan, or (f) the amide group of glutamine.

Removal of any carbohydrate moieties present on the polypeptide orantibody are, in some embodiments, accomplished chemically orenzymatically. Chemical deglycosylation involves exposure of theantibody to the compound trifluoromethanesulfonic acid, or an equivalentcompound. This treatment results in the cleavage of most or all sugarsexcept the linking sugar (N-acetylglucosamine or N-acetylgalactosamine),while leaving the antibody intact. Enzymatic cleavage of carbohydratemoieties on an antibody is achieved by the use of a variety of endo- andexo-glycosidases in some embodiments.

Another type of covalent modification comprises linking an antibody toone of a variety of nonproteinaceous polymers, e.g., polyethyleneglycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylatedsorbitol, polyoxyethylated glucose, polyoxyethylated glycerol,polyoxyalkylenes, or polysaccharide polymers such as dextran. Suchmethods are known in the art.

Affinity for binding a pre-determined polypeptide antigen, generally, ismodulated by introducing one or more mutations into the V regionframework, typically in areas adjacent to one or more CDRs and/or in oneor more framework regions. Typically, such mutations involve theintroduction of conservative amino acid substitutions that eitherdestroy or create the glycosylation site sequences but do notsubstantially affect the hydropathic structural properties of thepolypeptide. Typically, mutations that introduce a proline residue areavoided.

Effector Functions

Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;down-regulation of cell surface receptors (e.g., B cell receptor); and Bcell activation. Typically, the Fc-mediated functions involve binding ofthe Fc portion of the antibody by specialized receptor molecules, “Fcreceptors” or “FcR,” expressed by the cell whose function is to beaffected.

IgG is considered the most versatile immunoglobulin, because it carriesout all of the functions of immunoglobulin molecules in someembodiments. IgG is the major Ig in serum, and the only class of Ig thatcrosses the placenta. IgG also fixes complement, although the IgG4subclass does not. Macrophages, monocytes, polymorphonuclear leukocytes(PMNs), and some lymphocytes have receptors for the Fc region of IgG.Not all subclasses bind equally well: IgG2 and IgG4 do not bind to Fcreceptors. A consequence of binding to the Fc receptors on PMNs,monocytes, and macrophages is that the cell now internalizes the antigenbetter in some cases. IgG is an opsonin that enhances phagocytosis.Binding of IgG to Fc receptors on other types of cells results in theactivation of other functions.

In certain embodiments, the FcR is a native sequence human FcR.Moreover, a preferred FcR is one that binds an IgG antibody (a gamma(“γ”) receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are required for such killing. The primary cells for mediatingADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI,FcγRII, and FcγRIII. To assess ADCC activity of a molecule of interest,an in vitro ADCC assay is performed in some embodiments. Useful effectorcells for such assays include peripheral blood mononuclear cells (PBMC)and Natural Killer (NK) cells.

Alternatively, or additionally, in some embodiments, ADCC activity ofthe molecule of interest is assessed in vivo, e.g., in an animal model.

In some embodiments, the antibodies of the disclosure bind to a surfacemembrane protein of and are internalized by M2-like macrophages. Thisinternalization process is believed to be involved in the observedalteration of the functional immunosuppressive characteristics of thesecells, i.e., the differentiation of the cells from M2 status to subtlyactivated state, without killing them or inhibiting their proliferation.In some embodiments, upon internalization, the antibodies decrease theexpression of immunosuppressive soluble factors while increasingexpression of soluble factors that stimulate or promote the activity orproliferation of T cells, including CD4⁺ helper T cells and cytotoxiclymphocytes.

For certain therapeutic applications, the internalization process isemployed for purposes of killing or decreasing the activity orproliferation of a target cell that expresses a CD163 protein. Thenumber of antibody molecules internalized will be sufficient or adequateto kill a cell or inhibit its growth, especially a cancer cell.Depending on the potency of the antibody or antibody conjugate, in someinstances, the uptake of a single antibody molecule into the cell issufficient to kill the target cell to which the antibody binds. Forexample, certain toxins are highly potent in killing such thatinternalization of one molecule of the toxin conjugated to the antibodyis sufficient to kill the targeted cell.

In some embodiments, the antibody or antigen-binding fragment providedherein is conjugated or linked to a therapeutic moiety, an imaging ordetectable moiety, or an affinity tag. Methods for conjugating orlinking polypeptides are well known in the art. Associations (binding)between compounds and labels include any means known in the artincluding, but not limited to, covalent and non-covalent interactions,chemical conjugation, as well as recombinant techniques. An antibody orantigen-binding fragment thereof is conjugated to, or recombinantlyengineered with, an affinity tag (e.g., a purification tag), in someembodiments. Affinity tags such as, for example, poly-histidine (e.g.,His6(SEQ ID NO: 21)) tags are conventional in the art.

In some embodiments, the antibody or antigen-binding fragment furthercomprises a detectable moiety. Detections accomplished, for example, invitro, in vivo or ex vivo. In vitro assays for the detection and/ordetermination (quantification, qualification, etc.) of, e.g., huCD163protein expressed by macrophages using the antibodies or antigen-bindingfragments thereof include but are not limited to, for example, ELISAs,RIAs, and western blots. In some embodiments, in vitro detection,diagnosis, or monitoring of the antigen of the antibodies occurs byobtaining a sample (e.g., a blood sample) from a subject and testing thesample in, for example, a standard ELISA assay.

Also disclosed herein, in certain embodiments, are compositionscomprising an antibody as disclosed herein, and a carrier.

Pharmaceutical Compositions

Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising an antibody as disclosed herein and apharmaceutically acceptable excipient.

Such compositions are useful for in vitro or in vivo analysis or, in thecase of pharmaceutical compositions, for administration to a subject invivo or ex vivo for treating a subject with the disclosed antibodies.

In some embodiments, the excipient is a carrier, buffer, stabilizer orother suitable materials known to those skilled in the art. Suchmaterials should be non-toxic and should not interfere with the efficacyof the active ingredient. The precise nature of the carrier or othermaterial will depend on the route of administration.

Pharmaceutical formulations comprising an antibody or antigen-bindingfragment, identified by the methods described herein are prepared forstorage by mixing the protein having the desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizers(see, e.g., Remington's Pharmaceutical Sciences, 16^(th) edition, Osol,A. Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions in some embodiments. Acceptable carriers, or stabilizers arethose that are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® orpolyethylene glycol (PEG). In certain embodiments, the pharmaceuticalcomposition comprises the antibody at a concentration of between 5-200mg/mL; preferably between 10-100 mg/mL.

Acceptable carriers are physiologically acceptable to the administeredsubject and retain the therapeutic properties of the compounds with/inwhich it is administered. Acceptable carriers and their formulations areand generally described in, for example, Remington's PharmaceuticalSciences, supra. One exemplary carrier is physiological saline. Thephrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject compoundsfrom the administration site of one organ, or portion of the body, toanother organ, or portion of the body, or in an in vitro assay system.Each carrier is acceptable in the sense of being compatible with theother ingredients of the formulation and not injurious to a subject towhom it is administered. Nor should an acceptable carrier alter thespecific activity of the subject compounds.

In another embodiment, a pharmaceutical composition disclosed hereinfurther comprises an acceptable additive to improve the stability of thecompounds in composition and/or to control the release rate of thecomposition. Acceptable additives do not alter the specific activity ofthe subject compounds. Exemplary acceptable additives include, but arenot limited to, a sugar such as mannitol, sorbitol, glucose, xylitol,trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose,lactose, and mixtures thereof. Acceptable additives are combined withacceptable carriers and/or excipients such as dextrose in someembodiments. Alternatively, exemplary acceptable additives include, butare not limited to, a surfactant such as polysorbate 20 or polysorbate80 to increase stability of the peptide and decrease gelling of thesolution. In some embodiments, the surfactant is added to thecomposition in an amount of 0.01% to 5% of the solution. Addition ofsuch acceptable additives increases the stability and half-life of thecomposition in storage.

In one embodiment, a pharmaceutical composition disclosed hereincontains an isotonic buffer such as a phosphate, acetate, or TRIS bufferin combination with a tonicity agent such as a polyol, Sorbitol, sucroseor sodium chloride, which tonicifies and stabilizes. In someembodiments, a tonicity agent is present in the composition in an amountof about 5%.

In another embodiment, a pharmaceutical composition disclosed hereinincludes a surfactant such as to prevent aggregation and forstabilization at 0.01 to 0.02% wt/vol.

In another embodiment, the pH of a pharmaceutical composition disclosedherein ranges from 4.5-6.5 or 4.5-5.5.

In some embodiments, a pharmaceutical composition disclosed herein alsocontains more than one active compound as necessary for the indicationbeing treated, such as those with complementary activities that do notadversely affect each other. For example, a method of treatment furtherprovides an immunosuppressive agent. Such molecules are suitably presentin combination in amounts that are effective for the purpose intended.

In some embodiments, active ingredients are entrapped in microcapsuleprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxy methylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

Suspensions and crystal forms of antibodies are also contemplatedherein; methods to make suspensions and crystal forms are known to oneof skill in the art.

In some embodiments, a pharmaceutical composition disclosed herein issterile. In some embodiments, a pharmaceutical composition disclosedherein is sterilized by conventional, well known sterilizationtechniques. For example, sterilization is readily accomplished byfiltration through sterile filtration membranes. In some embodiments,the resulting solutions is packaged for use or filtered under asepticconditions and lyophilized, the lyophilized preparation being combinedwith a sterile solution prior to administration.

Freeze-drying is employed to stabilize polypeptides for long-termstorage, such as when a polypeptide is relatively unstable in liquidcompositions, in some embodiments.

In some embodiments, some excipients such as, for example, polyols(including mannitol, sorbitol, and glycerol); sugars (including glucoseand sucrose); and amino acids (including alanine, glycine, and glutamicacid), act as stabilizers for freeze-dried products. Polyols and sugarsare also used to protect polypeptides from freezing and drying-induceddamage and to enhance the stability during storage in the dried state insome embodiments. Sugars are, in some embodiments, effective in both thefreeze-drying process and during storage. Other classes of molecules,including mono- and disaccharides and polymers such as PVP, have alsobeen reported as stabilizers of lyophilized products.

For injection, in some embodiments, a pharmaceutical compositiondisclosed herein is a powder suitable for reconstitution with anappropriate solution as described above. Examples of these include, butare not limited to, freeze dried, rotary dried or spray dried powders,amorphous powders, granules, precipitates, or particulates. Forinjection, the compositions optionally contain stabilizers, pHmodifiers, surfactants, bioavailability modifiers and combinations ofthese.

Sustained-release preparations is prepared in some embodiments. Suitableexamples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (see, e.g., U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the Lupron Depot™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. In some embodiments, while encapsulated antibodies remain inthe body for a long time, they denature or aggregate as a result ofexposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategiesdevised for stabilization are, in some cases, dependent on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization is achieved, in some cases, by modifying sulfhydrylresidues, lyophilizing from acidic solutions, controlling moisturecontent, using appropriate additives, and developing specific polymermatrix compositions.

In some embodiments, a pharmaceutical composition disclosed herein isdesigned to be short-acting, fast-releasing, long-acting, orsustained-releasing as described herein. In one embodiment, apharmaceutical composition disclosed herein is formulated for controlledrelease or for slow release.

The pharmaceutical composition is administered, for example, byinjection, including, but not limited to, subcutaneous, intravitreal,intradermal, intravenous, intra-arterial, intraperitoneal,intracerobrospinal, or intramuscular injection. Excipients and carriersfor use in formulation of compositions for each type of injection arecontemplated herein. The following descriptions are by example only andare not meant to limit the scope of the compositions. Compositions forinjection include, but are not limited to, aqueous solutions (wherewater soluble) or dispersions, as well as sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). In someembodiments, the carrier is a solvent or dispersion medium containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. Fluidity is maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants.Antibacterial and antifungal agents include, for example, parabens,chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents,for example, sugars, polyalcohols such as mannitol, sorbitol, and sodiumchloride is included in the composition in some embodiments. In someembodiments, the resulting solutions are packaged for use as is, orlyophilized; the lyophilized preparation is later be combined with asterile solution prior to administration in some embodiments. Forintravenous, injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicity,and stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, and Lactated Ringer'sInjection. Preservatives, stabilizers, buffers, antioxidants, and/orother additives are included, as needed, in some embodiments. Sterileinjectable solutions are prepared by incorporating an active ingredientin the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization, in some embodiments. Generally, dispersions areprepared by incorporating the active ingredient into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Compositions are conventionally administered intravenously in someembodiments, such as by injection of a unit dose, for example. Forinjection, in some embodiments, an active ingredient is in the form of aparenterally acceptable aqueous solution which is substantiallypyrogen-free and has suitable pH, isotonicity, and stability. In someembodiments, one prepares suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilizers, buffers,antioxidants, and/or other additives are included, as required, in someembodiments. Additionally, compositions are administered viaaerosolization in some embodiments. (Lahn et al., Int Arch AllergyImmunol 134:49-55 (2004)).

For parenteral administration, the antibodies are formulated in a unitdosage injectable form (solution, suspension, emulsion) in associationwith a pharmaceutically acceptable, parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Nonaqueous vehicles such as fixed oils and ethyloleate are also used. Liposomes are used as carriers. The vehiclecontains minor amounts of additives such as substances that enhanceisotonicity and chemical stability, e.g., buffers and preservatives. Theantibodies are typically formulated in such vehicles at concentrationsof about 1 mg/mL to 10 mg/mL.

In one embodiment, a pharmaceutical composition disclosed herein islyophilized, for example, to increase shelf-life in storage. When thecompositions are considered for use in medicaments or any of the methodsprovided herein, in some embodiments, it is contemplated that thecomposition are substantially free of pyrogens such that the compositionwill not cause an inflammatory reaction or an unsafe allergic reactionwhen administered to a human subject. Testing compositions for pyrogensand preparing compositions substantially free of pyrogens are wellunderstood to one or ordinary skill of the art and are accomplishedusing commercially available kits in some embodiments.

In some embodiments, acceptable carriers contain a compound thatstabilizes, increases or delays absorption or clearance. Such compoundsinclude, for example, carbohydrates, such as glucose, sucrose, ordextrans; low molecular weight proteins; compositions that reduce theclearance or hydrolysis of peptides; or excipients or other stabilizersand/or buffers. Agents that delay absorption include, for example,aluminum monostearate and gelatin. In some embodiments, detergents alsobe used to stabilize or to increase or decrease the absorption of thepharmaceutical composition, including liposomal carriers. To protectfrom digestion the compound, in some embodiments, is complexed with acomposition to render it resistant to acidic and enzymatic hydrolysis,or the compound is, in some embodiments, complexed in an appropriatelyresistant carrier such as a liposome. Means of protecting compounds fromdigestion are known in the art.

The compositions are administered, in some embodiments, in a mannercompatible with the dosage formulation, and in a therapeuticallyeffective amount. The quantity to be administered depends on the subjectto be treated, capacity of the subject's immune system to utilize theactive ingredient, and degree of binding capacity desired. Preciseamounts of active ingredient required to be administered depend on thejudgment of the practitioner and are peculiar to each individual.Suitable regimes for initial administration and booster shots are alsovariable, but are typified by an initial administration followed byrepeated doses at one or more hour intervals by a subsequent injectionor other administration. Alternatively, continuous intravenous infusionthat is sufficient to maintain concentrations in the blood arecontemplated.

In some embodiments, the disclosure provides a use of the compositionsdescribed herein to make a medicament for treating a condition, disease,or disorder described herein. In some embodiments, medicaments areformulated based on the physical characteristics of the subject needingtreatment, and are formulated in single or multiple formulations basedon the stage of the condition, disease or disorder. Medicaments arepackaged in a suitable package with appropriate labels for thedistribution to hospitals and clinics in which the label is for theindication of treating a subject having a disease described herein insome embodiments. Medicaments are packaged as a single or multiple unitsin some embodiments. Instructions for the dosage and administration ofthe compositions are included with the packages as described below insome embodiments. The disclosure is further directed to medicamentscomprising an antibody or antigen-binding fragment thereof describedherein and a pharmaceutically acceptable carrier.

In some embodiments, a composition (an antibody or an antigen-bindingfragment described herein) is administered alone or in combination witha second composition either simultaneously or sequentially dependentupon the condition to be treated. In one embodiment, a secondtherapeutic treatment is an anticancer therapy or an anticancertherapeutic. When two or more compositions are administered, thecompositions are, for example, administered in combination (eithersequentially or simultaneously). A composition is administered in asingle dose or multiple doses in some embodiments.

In some embodiments, when formulated for administration to humansubjects, the compositions are formulated to be free of pyrogens.Testing compositions for pyrogens and preparing pharmaceuticalcompositions free of pyrogens are well understood to one of ordinaryskill in the art.

Antibodies, or antigen-binding fragments thereof, are formulated for anysuitable route of administration to a subject including, but not limitedto injection, in some embodiments. Injection includes, for example,subcutaneous, peritoneal, intravenous injection, intramuscularinjection, or spinal injection into the cerebrospinal fluid (CSF). Insome embodiments, administration are in one, two, three, four, five,six, seven, or more injection sites. In one embodiment, administrationis via six injection sites.

For in vivo applications, contacting occurs, for example, viaadministration of a composition (such as are described herein) to asubject by any suitable means. An antibody described herein, in someembodiments, is administered by any suitable means, either systemicallyor locally, including via parenteral, subcutaneous, intraperitoneal,intracerobrospinal, intrapulmonary, and intranasal administration, and,if desired for local treatment, intralesional administration. Parenteralroutes include, for example, intravenous, intraarterial,intraperitoneal, epidural, intramuscular, and intrathecaladministration. Such administration, in some embodiments, is as a bolus,continuous infusion, or pulse infusion. In some embodiments,compositions are administered by injection depending in part on whetherthe administration is brief or chronic. Other modes of administrationmethods are contemplated, including topical, particularly transdermal,transmucosal, rectal, oral or local administration e.g., through acatheter placed close to the desired site.

Methods of Treatment and Use

Disclosed herein, in certain embodiments, are methods of treating acancer in an individual in need thereof, comprising administering to theindividual an antibody disclosed herein. In some embodiments, thedisclosure provides a use of an antibody as described herein, for themanufacture of a medicament for treating cancer in a human subject. Insome embodiments, the antibody specifically binds to a CD163 proteinexpressed on human tumor associated macrophages and reduces expressionof at least one of CD16, CD64, TLR2, or Siglec-15 by the macrophages.

Disclosed herein, in certain embodiments, are methods of modulatingimmune activity in a subject in need thereof, comprising administeringto said subject an antibody described herein. In some embodiments, theantibody specifically binds to a CD163 protein expressed on human tumorassociated macrophages and reduces expression of at least one of CD16,CD64, TLR2, or Siglec-15 by the macrophages.

Disclosed herein, in certain embodiments, are methods of treating asubject with pathologically or inappropriately elevated levels of M2macrophages (e.g., inappropriately elevated relative to the level usefulfor promoting immune-mediated tumor cell killing in the subject),comprising administering to said subject an antibody described herein.In some embodiments, the antibody specifically binds to a CD163 proteinexpressed on human tumor associated macrophages and reduces expressionof at least one of CD16, CD64, TLR2, or Siglec-15 by the macrophages.

In some embodiments, the antibody comprises at least one of a lightchain CDR1 having an amino acid sequence at least about 70% identical toan amino acid sequence set forth as SEQ ID NO: 1, a light chain CDR2having an amino acid sequence at least about 70% identical to an aminoacid sequence set forth as SEQ ID NO: 2, and a light chain CDR3 havingan amino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 3. In some embodiments, the antibodycomprises at least one of a light chain a light chain CDR1 having anamino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence set forth as SEQ ID NO: 1, a lightchain CDR2 having an amino acid sequence at least about 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to an amino acid sequence set forth asSEQ ID NO: 2, and a light chain CDR3 having an amino acid sequence atleast about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an aminoacid sequence set forth as SEQ ID NO: 3. In some embodiments, theantibody comprises at least one of a light chain CDR1 having an aminoacid sequence 100% identical to an amino acid sequence set forth as SEQID NO: 1, a light chain CDR2 having an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 2, and alight chain CDR3 having an amino acid sequence 100% identical to anamino acid sequence set forth as SEQ ID NO: 3.

In some embodiments, the antibody comprises at least one of a heavychain CDR1 having an amino acid sequence at least about 70% identical toan amino acid sequence set forth as SEQ ID NO: 4, a heavy chain CDR2having an amino acid sequence at least about 70% identical to an aminoacid sequence set forth as SEQ ID NO: 5, a heavy chain CDR3 having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 6. In some embodiments, the antibodycomprises at least one of a heavy chain CDR1 having an amino acidsequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto an amino acid sequence set forth as SEQ ID NO: 4, a heavy chain CDR2having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence set forth as SEQ ID NO:5, a heavy chain CDR3 having an amino acid sequence at least about 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence setforth as SEQ ID NO: 6. In some embodiments, the antibody comprises atleast one of a heavy chain CDR1 having an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 4, a heavychain CDR2 having an amino acid sequence 100% identical to an amino acidsequence set forth as SEQ ID NO: 5, a heavy chain CDR3 having an aminoacid sequence 100% identical to an amino acid sequence set forth as SEQID NO: 6.

In some embodiments, the antibody comprises at least one of a lightchain CDR1 having an amino acid sequence at least about 70% identical toan amino acid sequence set forth as SEQ ID NO: 1, a light chain CDR2having an amino acid sequence at least about 70% identical to an aminoacid sequence set forth as SEQ ID NO: 2, a light chain CDR3 having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 3, a heavy chain CDR1 having an aminoacid sequence at least about 70% identical to an amino acid sequence setforth as SEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequenceat least about 70% identical to an amino acid sequence set forth as SEQID NO: 5, and a heavy chain CDR3 having an amino acid sequence at leastabout 70% identical to an amino acid sequence set forth as SEQ ID NO: 6.In some embodiments, the antibody comprises at least one of a lightchain CDR1 having an amino acid sequence at least about 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to an amino acid sequence set forth asSEQ ID NO: 1, a light chain CDR2 having an amino acid sequence at leastabout 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acidsequence set forth as SEQ ID NO: 2, a light chain CDR3 having an aminoacid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence set forth as SEQ ID NO: 3, a heavychain CDR1 having an amino acid sequence at least about 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to an amino acid sequence set forth asSEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequence at leastabout 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acidsequence set forth as SEQ ID NO: 5, and a heavy chain CDR3 having anamino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence set forth as SEQ ID NO: 6. In someembodiments, the antibody comprises at least one of a light chain CDR1having an amino acid sequence 100% identical to an amino acid sequenceset forth as SEQ ID NO: 1, a light chain CDR2 having an amino acidsequence 100% identical to an amino acid sequence set forth as SEQ IDNO: 2, a light chain CDR3 having an amino acid sequence 100% identicalto an amino acid sequence set forth as SEQ ID NO: 3, a heavy chain CDR1having an amino acid sequence 100% identical to an amino acid sequenceset forth as SEQ ID NO: 4, a heavy chain CDR2 having an amino acidsequence 100% identical to an amino acid sequence set forth as SEQ IDNO: 5, and a heavy chain CDR3 having an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 6.

In some embodiments, the antibody comprises a light chain variabledomain (VL) having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 7. In some embodimentsthe VL has an amino acid sequence at least about 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to an amino acid sequence set forth as SEQ IDNO: 7. In some embodiments, the VL has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 7.

In some embodiments, the antibody comprises a heavy chain variabledomain (VH) having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 8. In some embodimentsthe VH has an amino acid sequence at least about 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to an amino acid sequence set forth as SEQ IDNO: 8. In some embodiments, the VH has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 8.

In some embodiments, the antibody comprises a light chain variabledomain (VL) having an amino acid sequence at least about 70% identicalto an amino acid sequence set forth as SEQ ID NO: 7 and a heavy chainvariable domain (VH) having an amino acid sequence at least about 70%identical to an amino acid sequence set forth as SEQ ID NO: 8. In someembodiments the VL has an amino acid sequence at least about 75%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forthas SEQ ID NO: 7 and the VH has an amino acid sequence at least about75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequenceset forth as SEQ ID NO: 8. In some embodiments, the VL has an amino acidsequence 100% identical to an amino acid sequence set forth as SEQ IDNO: 7 and the VH has an amino acid sequence 100% identical to an aminoacid sequence set forth as SEQ ID NO: 8.

In some embodiments, the antibody comprises a light chain having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 9. In some embodiments the light chainhas an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to an amino acid sequence set forth as SEQ ID NO: 9. Insome embodiments, the light chain has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 9.

In some embodiments, the antibody comprises a heavy chain having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 10. In some embodiments the heavy chainhas an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to an amino acid sequence set forth as SEQ ID NO: 10. Insome embodiments, the heavy chain has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 10.

In some embodiments, the antibody comprises a heavy chain having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 11. In some embodiments the heavy chainhas an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to an amino acid sequence set forth as SEQ ID NO: 11. Insome embodiments, the heavy chain has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 11.

In some embodiments, the antibody comprises a heavy chain having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 12. In some embodiments the heavy chainhas an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to an amino acid sequence set forth as SEQ ID NO: 12. Insome embodiments, the heavy chain has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 12.

In some embodiments, the antibody comprises a heavy chain having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 13. In some embodiments the heavy chainhas an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to an amino acid sequence set forth as SEQ ID NO: 13. Insome embodiments, the heavy chain has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 13.

In some embodiments, the antibody comprises a light chain having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 9 and a heavy chain having an aminoacid sequence at least about 70% identical to an amino acid sequence setforth as SEQ ID NO: 10. In some embodiments the light chain has an aminoacid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence set forth as SEQ ID NO: 9 and theheavy chain has an amino acid sequence at least about 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to an amino acid sequence set forth asSEQ ID NO: 10. In some embodiments, the light chain has an amino acidsequence 100% identical to an amino acid sequence set forth as SEQ IDNO: 9 and the heavy chain has an amino acid sequence 100% identical toan amino acid sequence set forth as SEQ ID NO: 10.

In some embodiments, the antibody comprises a light chain having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 9 and a heavy chain having an aminoacid sequence at least about 70% identical to an amino acid sequence setforth as SEQ ID NO: 11. In some embodiments the light chain has an aminoacid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence set forth as SEQ ID NO: 9 and theheavy chain has an amino acid sequence at least about 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to an amino acid sequence set forth asSEQ ID NO: 11. In some embodiments, the light chain has an amino acidsequence 100% identical to an amino acid sequence set forth as SEQ IDNO: 9 and the heavy chain has an amino acid sequence 100% identical toan amino acid sequence set forth as SEQ ID NO: 11.

In some embodiments, the antibody comprises a light chain having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 9 and a heavy chain having an aminoacid sequence at least about 70% identical to an amino acid sequence setforth as SEQ ID NO: 12. In some embodiments the light chain has an aminoacid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence set forth as SEQ ID NO: 9 and theheavy chain has an amino acid sequence at least about 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to an amino acid sequence set forth asSEQ ID NO: 12. In some embodiments, the light chain has an amino acidsequence 100% identical to an amino acid sequence set forth as SEQ IDNO: 9 and the heavy chain has an amino acid sequence 100% identical toan amino acid sequence set forth as SEQ ID NO: 12.

In some embodiments, the antibody comprises a light chain having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 9 and a heavy chain having an aminoacid sequence at least about 70% identical to an amino acid sequence setforth as SEQ ID NO: 13. In some embodiments the light chain has an aminoacid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence set forth as SEQ ID NO: 9 and theheavy chain has an amino acid sequence at least about 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to an amino acid sequence set forth asSEQ ID NO: 13. In some embodiments, the light chain has an amino acidsequence 100% identical to an amino acid sequence set forth as SEQ IDNO: 9 and the heavy chain has an amino acid sequence 100% identical toan amino acid sequence set forth as SEQ ID NO: 13.

In some embodiments, the antibody comprises a light chain having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 14. In some embodiments the light chainhas an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to an amino acid sequence set forth as SEQ ID NO: 14. Insome embodiments, the light chain has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 14.

In some embodiments, the antibody comprises a heavy chain having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 15. In some embodiments the heavy chainhas an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to an amino acid sequence set forth as SEQ ID NO: 15. Insome embodiments, the heavy chain has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 15.

In some embodiments, the antibody comprises a heavy chain having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 16. In some embodiments the heavy chainhas an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to an amino acid sequence set forth as SEQ ID NO: 16. Insome embodiments, the heavy chain has an amino acid sequence 100%identical to an amino acid sequence set forth as SEQ ID NO: 16.

Also In some embodiments, the antibody comprises a light chain having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 14 and a heavy chain having an aminoacid sequence at least about 70% identical to an amino acid sequence setforth as SEQ ID NO: 15. In some embodiments the light chain has an aminoacid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence set forth as SEQ ID NO: 14 and theheavy chain has an amino acid sequence at least about 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to an amino acid sequence set forth asSEQ ID NO: 15. In some embodiments, the light chain has an amino acidsequence 100% identical to an amino acid sequence set forth as SEQ IDNO: 14 and the heavy chain has an amino acid sequence 100% identical toan amino acid sequence set forth as SEQ ID NO: 15.

In some embodiments, the antibody comprises a light chain having anamino acid sequence at least about 70% identical to an amino acidsequence set forth as SEQ ID NO: 14 and a heavy chain having an aminoacid sequence at least about 70% identical to an amino acid sequence setforth as SEQ ID NO: 16. In some embodiments the light chain has an aminoacid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence set forth as SEQ ID NO: 14 and theheavy chain has an amino acid sequence at least about 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to an amino acid sequence set forth asSEQ ID NO: 16. In some embodiments, the light chain has an amino acidsequence 100% identical to an amino acid sequence set forth as SEQ IDNO: 14 and the heavy chain has an amino acid sequence 100% identical toan amino acid sequence set forth as SEQ ID NO: 16.

In some embodiments, the disclosure provides a use of an antibody asdescribed herein, for the manufacture of a medicament that reducesimmunosuppression by tumor-associated macrophages in a human subjecthaving a cancer.

In some embodiments, the disclosure provides a use of an antibody asdescribed herein, for the manufacture of a medicament that promotes Tcell-mediated tumor cell killing in a human subject having a cancer.

In some embodiments, the disclosure provides a method of treating ahuman subject having a cancer, comprising administering to the subject atherapeutically effective amount of an antibody as described herein,whereby immunosuppression by tumor-associated macrophages in the subjectis reduced.

In some embodiments, the disclosure provides a method of treating ahuman subject having a cancer, comprising administering to the subject atherapeutically effective amount of an antibody as described herein,whereby T cell-mediated tumor cell killing in the subject is increased.

Disclosed herein, in certain embodiments, are methods of functionallyreorienting tumor-associated macrophages to reduce immunosuppression ina patient having cancer, comprising administering to the patient anamount of a pharmaceutical composition comprising an antibody asdescribed herein that is effective to improve CD4⁺ or CD8⁺ T cellactivity or proliferation in the tumor microenvironment.

Disclosed herein, in certain embodiments, are methods of promotinglymphocyte-mediated tumor cell killing in a human subject in needthereof, comprising administering to the subject an effective amount ofa pharmaceutical composition comprising an antibody as described herein.

Disclosed herein, in certain embodiments, are methods of modulating anactivity of a tumor-associated macrophage in a tumor microenvironment,the method comprising contacting the tumor-associated macrophage with anantibody disclosed herein, wherein the method results in at least one ofthe following effects:

-   -   (a) reduced expression of at least one marker by the human        macrophage, wherein the at least one marker is CD16, CD64, TLR2,        or Siglec-15;    -   (b) internalization of the antibody by the human macrophage;    -   (c) activation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof;    -   (d) proliferation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof; and    -   (e) promotion of tumor cell killing in a tumor microenvironment.

Disclosed herein, in certain embodiments, are methods of modulating anactivity of a tumor-associated macrophage in a tumor microenvironment,the method comprising contacting the tumor-associated macrophage with anantibody disclosed herein, wherein the method results in at least two ofthe following effects:

-   -   (a) reduced expression of at least one marker by the human        macrophage, wherein the at least one marker is CD16, CD64, TLR2,        or Siglec-15;    -   (b) internalization of the antibody by the human macrophage;    -   (c) activation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof;    -   (d) proliferation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof; and    -   (e) promotion of tumor cell killing in a tumor microenvironment.

Disclosed herein, in certain embodiments, are methods of modulating anactivity of a tumor-associated macrophage in a tumor microenvironment,the method comprising contacting the tumor-associated macrophage with anantibody disclosed herein, wherein the method results in at least threeof the following effects:

-   -   (a) reduced expression of at least one marker by the human        macrophage, wherein the at least one marker is CD16, CD64, TLR2,        or Siglec-15;    -   (b) internalization of the antibody by the human macrophage;    -   (c) activation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof;    -   (d) proliferation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof; and    -   (e) promotion of tumor cell killing in a tumor microenvironment.

Disclosed herein, in certain embodiments, are methods of modulating anactivity of a tumor-associated macrophage in a tumor microenvironment,the method comprising contacting the tumor-associated macrophage with anantibody disclosed herein, wherein the method results in at least fourof the following effects:

-   -   (a) reduced expression of at least one marker by the human        macrophage, wherein the at least one marker is CD16, CD64, TLR2,        or Siglec-15;    -   (b) internalization of the antibody by the human macrophage;    -   (c) activation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof;    -   (d) proliferation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof; and    -   (e) promotion of tumor cell killing in a tumor microenvironment.

Disclosed herein, in certain embodiments, are methods of modulating anactivity of a tumor-associated macrophage in a tumor microenvironment,the method comprising contacting the tumor-associated macrophage with anantibody disclosed herein, wherein the method results in at least fiveof the following effects:

-   -   (a) reduced expression of at least one marker by the human        macrophage, wherein the at least one marker is CD16, CD64, TLR2,        or Siglec-15;    -   (b) internalization of the antibody by the human macrophage;    -   (c) activation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof;    -   (d) proliferation of a CD4⁺ T cell, CD8⁺ T cell, NK cell, or any        combination thereof, and    -   (e) promotion of tumor cell killing in a tumor microenvironment.

In some embodiments, a method disclosed herein reduces myeloid cellsuppression of CD8 T cell activation and proliferation.

M2 to M1 macrophage polarization refers to the process by which a M2 orM2-like macrophage of the present disclosure is altered such that theresulting macrophage has certain functional or phenotypic attributesassociated with M1 or M1-like macrophages. In some embodiments, an M2 orM2-like macrophage is polarized when it no longer expresses CD163.

In some embodiments, the antibodies reduce myeloid cell suppression ofCD19-CD3 BiTE-mediated killing of Raji cells by CD8 T cells.

In some embodiments, the antibodies reduce myeloid cell suppression ofCAR T-cell-mediated killing of cancer cells.

In some embodiments, the antibodies reduce myeloid cell suppression ofNK cell-mediated killing of cancer cells by ADCC.

Any of the methods disclosed herein, in some instances, further compriseadministering to said subject an additional anticancer therapy.Anticancer therapies include, but are not limited to, surgical therapy,chemotherapy, radiation therapy, cryotherapy, hormonal therapy,immunotherapy, and cytokine therapy, and combinations thereof. In oneembodiment, the antibody or antigen-binding fragment thereof and theanticancer therapy are administered concurrently or sequentially.

In some embodiments, the additional anticancer therapy is animmunotherapy. In some embodiments, the immunotherapy is a compositioncomprising a checkpoint inhibitor. In some embodiments, the additionalanticancer therapy is an immune checkpoint inhibitor.

In some embodiments, the disclosure provides an in vitro or ex vivomethod for identifying huCD163-expressing macrophages in a cell samplesuspected of comprising huCD163-expressing macrophages, comprisingcontacting the cell sample with an antibody as described herein andmeasuring binding to cells in the sample, a positive signal indicatingthe presence of huCD163-expressing macrophages in the sample.

In some embodiments, the disclosure provides a method for identifyinghuman M2c macrophages in a cell sample, which comprises contacting acell sample comprising blood cells suspected of comprising human M2cmacrophages with an antibody as described herein and measuring bindingto cells in the sample, a positive signal indicating the presence ofhuman M2c macrophages in the sample. In some embodiments, the cellsample comprises cells obtained from a human subject. In someembodiments, the cell sample comprises cultured cells. In someembodiments, the methods for detecting M2 cells in a sample includeusing an antibody or fragment that is not internalized by the cells uponbinding, but remains bound to the exterior of the cells to facilitatedetection.

In some embodiments, the disclosure provides a method for identifyinghuCD163-expressing cancer cells in a cell sample, which comprisescontacting a cell sample comprising cells suspected of comprisinghuCD163-expressing cancer cells with an antibody as described herein andmeasuring binding to cells in the sample, a positive signal indicatingthe presence of huCD163-expressing cancer cells in the sample. In someembodiments, the cell sample comprises cells obtained from a humansubject. In some embodiments, the cell sample comprises cultured cells.

In some embodiments, the disclosure provides a method of modulatingexpression of cell surface markers on M2c macrophages. In someembodiments, the expression of at least one of CD16 (FcγRIIIa), CD64(FcγRI), Siglec-15, and TLR2 is decreased. In some embodiments, theexpression of CD16, CD64, Siglec-15, and TLR2 are decreased.

In some embodiments, the disclosure provides a method of reducingmyeloid cell suppression of T cell activation. In some embodiments themethod induces increased IL-2 production by T cells. In someembodiments, the disclosure provides a method for increasing Th1 cellproliferation. In some embodiments, the disclosure provides a method forincreasing the proportion of Th1 cells in proliferating T cells. In eachof these aspects, the methods comprise administering an antibody asdescribed herein in vivo, or contacting the antibody with a compleximmune cell system in vitro or ex vivo, in which macrophages andeffector T cells, and optional tumor target cells, are allowed tointeract in ways that recapitulate or model in vivo interactions.

In some embodiments, the disclosure provides a method of reducingmyeloid cell suppression of T cell proliferation. In some embodiments,the disclosure provides a method for increasing CD4⁺ cell proliferationor CD8⁺ T cell proliferation, or both.

In some embodiments, the disclosure provides a method for increasing Tcell expression of at least one of CD69, ICOS, OX40, PD-1, LAG3, andCTLA4 in a patient in need thereof. In some embodiments the methodincreases expression of at least one of CD69, ICOS, OX40, PD-1, LAG3,and CTLA4 by CD4⁺ T cells. In some embodiments the method increasesexpression of at least one of ICOS, OX40, PD-1, LAG3, and CTLA4 by CD8⁺T cells.

In some embodiments, the disclosure provides a method for increasingcancer cell killing. In some embodiments cancer cell killing bycytotoxic lymphocytes (CTL) is increased. In some embodiments, Tcell-mediated killing of MHC-mismatched cancer cells is increased.

In some embodiments, the disclosure provides a method for contactingmyeloid cells with an antibody described herein, to reducemacrophage-mediated suppression of CD8⁺ T cell activation or CD8⁺ T cellproliferation. In some embodiments, the antibody is contacted withmyeloid cells comprising M0 macrophages. In some embodiments, theantibody is contacted with myeloid cells comprising M2 macrophages. Insome embodiments, the antibody is contacted with myeloid cellscomprising M0 and M2 macrophages.

In some embodiments the disclosure provides a method of treating a humanpatient having lung cancer, comprising administering an effective amountof an antibody as described herein to the patient. In some embodiments,the lung cancer is a carcinoma or an adenocarcinoma. In someembodiments, the lung cancer is non-small cell lung cancer.

An effective response of the present disclosure is achieved when thesubject experiences partial or total alleviation or reduction of signsor symptoms of illness and, in the case of the treatment of cancer,specifically includes, without limitation, cure, remission, prolongationof survival, or other objective responses. In some embodiments, theexpected progression-free survival times are measured in months toyears, depending on prognostic factors including the number of relapses,stage of disease, and other factors. Prolonging survival includeswithout limitation times of at least 1 month (mo.), about at least 2mos., about at least 3 mos., about at least 4 mos., about at least 6mos., about at least 1 year, about at least 2 years, about at least 3years, etc. Overall survival is also measured, for example, in months toyears. Alternatively, an effective response, in some embodiments, isthat a subject's symptoms remain static. Further indications oftreatment of indications are described in more detail below.

In some embodiments, administration of a therapeutic agent in aprophylactic method occurs prior to the manifestation of symptoms of anundesired disease or disorder, such that the disease or disorder isprevented or, alternatively, delayed in its progression. Thus, when usedin conjunction with prophylactic methods, the term “therapeuticallyeffective” means that, after treatment, a smaller number of subjects (onaverage) develop the undesired disease or disorder or progress inseverity of symptoms.

In some embodiments, amounts of the active ingredients in thecompositions, the composition formulation, and the mode ofadministration, are among the factors that are varied to provide anamount of the active ingredient that is effective to achieve the desiredtherapeutic response for each subject, without being unduly toxic to thesubject. The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound employed, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular composition employed, the age, sex,weight, condition, general health, diet and prior medical history of thesubject being treated, and like factors well known in the medical arts.

In some embodiments, the antibodies and antigen-binding fragmentsdescribed herein are administered to a subject in various dosing amountsand over various time frames. Non-limiting doses include about 0.01mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg,about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80mg/kg, about 90 mg/kg, about 100 mg/kg, about 125 mg/kg, about 150mg/kg, about 175 mg/kg, about 200 mg/kg, or any integer in between.Additionally, the dose(s) of an antibody or antigen-binding fragment areadministered, in some embodiments, twice a week, weekly, every twoweeks, every three weeks, every 4 weeks, every 6 weeks, every 8 weeks,every 12 weeks, or any combination of weeks therein. Dosing cycles arealso contemplated such as, for example, administering antibodies orantigen-binding fragments thereof once or twice a week for 4 weeks,followed by two weeks without therapy. Additional dosing cyclesincluding, for example, different combinations of the doses and weeklycycles described herein are also contemplated within the disclosure.

Therapeutically effective amounts of a composition, in some embodiments,varies and depends on the severity of the disease and the weight andgeneral state of the subject being treated, but generally range fromabout 1.0 μg/kg to about 100 mg/kg body weight, or about 10 μg/kg toabout 30 mg/kg, or about 0.1 mg/kg to about 10 mg/kg or about 1 mg/kg toabout 10 mg/kg per application. Administration can be daily, onalternating days, weekly, twice a month, monthly or more or lessfrequently, as necessary depending on the response to the disorder orcondition and the subject's tolerance of the therapy. In someembodiments, maintenance dosages over a longer period of time, such as4, 5, 6, 7, 8, 10, or 12 weeks or longer is needed until a desiredsuppression of disorder symptoms occurs, and dosages are adjusted asnecessary. The progress of this therapy is easily monitored byconventional techniques and assays.

In some embodiments, the antibody of the disclosure is administeredintravenously in a physiological solution at a dose ranging between 0.01mg/kg to 100 mg/kg at a frequency ranging from daily to weekly tomonthly (e.g., every day, every other day, every third day, or 2, 3, 4,5, or 6 times per week), preferably a dose ranging from 0.1 to 45 mg/kg,0.1 to 15 mg/kg or 0.1 to 10 mg/kg at a frequency of 2 or 3 times perweek, or up to 45 mg/kg once a month.

A response is achieved when the subject experiences partial or totalalleviation, or reduction of signs or symptoms of illness, andspecifically includes, without limitation, prolongation of survival. Theexpected progression-free survival times are measured, for example, inmonths to years, depending on prognostic factors including the number ofrelapses, stage of disease, and other factors. Prolonging survivalincludes without limitation times of at least 1 month (mo), about atleast 2 months (mos.), about at least 3 mos., about at least 4 mos.,about at least 6 mos., about at least 1 year, about at least 2 years,about at least 3 years, or more. Overall survival is also be measured inmonths to years in some embodiments. The subject's symptoms remainstatic or decrease in some embodiments.

A physician or veterinarian having ordinary skill in the art, in somecases, readily determines and prescribes the effective amount (ED₅₀) ofthe composition required. For example, the physician or veterinariancould start doses of the compounds employed in the composition at levelslower than that required to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.Alternatively, a dose remains constant in some embodiments.

In some embodiments, other antibodies, small molecule therapeutics,and/or other agents are combined in separate compositions forsimultaneous or sequential administration. In one embodiment,simultaneous administration comprises one or more compositions that areadministered at the same time, or within 30 minutes of each other. Insome embodiments, administration occurs at the same or different sites.

Toxicity and therapeutic efficacy of such ingredient are, in someembodiments, determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., for determining the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). In someembodiments, the dose ratio between toxic and therapeutic effects is thetherapeutic index and it are expressed as the ratio LD₅₀/ED₅₀. Whilecompounds that exhibit toxic side effects are used in some embodiments,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue to minimize potential damage tohealthy cells and, thereby, reduce side effects.

Data obtained from cell culture assays and animal studies is used informulating a range of dosage for use in humans in some embodiments. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. In someembodiments, the dosage varies within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the disclosure, the therapeuticallyeffective dose is estimated initially from cell culture assays in someembodiments. In some embodiments, a dose is formulated in animal modelsto achieve a circulating plasma concentration arrange that includes theIC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition) as determined in cell culture. Levels in plasmaare measured, for example, by high performance liquid chromatography.Such information is, in some cases, used to more accurately determineuseful doses in humans.

In some embodiments the disclosure provides a method of treating apatient having a cancer, comprising administering to the patient atherapeutically effective amount of an antibody as described herein andfurther comprising treating the subject with an anticancer therapyselected from surgical therapy, chemotherapy, radiation therapy,cryotherapy, hormonal therapy, immunotherapy, or cytokine therapy. Insome embodiments, the antibody, or antigen-binding fragment thereof, andanother anticancer therapy are administered concurrently orsequentially.

Diagnostic Products and Methods

In some embodiments, disclosed herein, are methods of detection of ahuCD163 protein or M2 macrophages in a sample or a subject to assess atreatment state of a patient or diagnose a disease or disorderassociated or correlated with the activity of M2 macrophages or TAMs,such as those diseases and disorders described herein.

In the in vivo detection, diagnosis or monitoring of soluble huCD163protein, expression of a huCD163 protein by cells or tissues, orpresence or activity of M2 macrophages, a subject is administered anantibody or antigen-binding fragment as described herein, which antibodyor antigen-binding fragment is bound to a detectable moiety. Thedetectable moiety is visualized, in some embodiments, usingart-recognized methods such as, but not limited to, magnetic resonanceimaging (MRI), fluorescence, radioimaging, light sources supplied byendoscopes, laparoscopes, or intravascular catheter (i.e., via detectionof photoactive agents), photoscanning, positron emission tomography(PET) scanning, whole body nuclear magnetic resonance (NMR),radioscintigraphy, single photon emission computed tomography (SPECT),targeted near infrared region (NIR) scanning, X-ray, ultrasound. Labelsfor detecting compounds using such methods are also known in the art.Visualization of the detectable moiety allows, in some embodiments, fordetection, diagnosis, and/or monitoring of a condition or diseaseassociated with M2 macrophage activity or activity of another cell thatexpresses a huCD163 protein. Additional diagnostic assays that utilizeantibodies specific to the desired target protein, i.e., a huCD163protein, are known in the art and are also contemplated herein.

For in vitro detection methods, samples to be obtained from a subjectinclude, but are not limited to, blood, tissue biopsy samples, and fluidtherefrom.

Thus, the disclosure provides antibodies and antigen-binding fragmentsthereof that are useful for detecting or diagnosing levels of M2macrophages or TAM macrophages associated with a disease or disorder,potentially indicating need for therapeutic treatment. In otherembodiments the antibody further comprises a second agent. Such anagent, in some embodiments, is a molecule or moiety such as, forexample, a reporter molecule or a detectable label. Detectablelabels/moieties for such detection methods are known in the art and aredescribed in more detail below. Reporter molecules are any moiety whichare detected using an assay, for example. Non-limiting examples ofreporter molecules which have been conjugated to polypeptides includeenzymes, radiolabels, haptens, fluorescent labels, phosphorescentmolecules, chemiluminescent molecules, chromophores, luminescentmolecules, photoaffinity molecules, colored particles or ligands, suchas biotin. In some embodiments, detectable labels include compoundsand/or elements that are detected due to their specific functionalproperties, and/or chemical characteristics, the use of which allows thepolypeptide to which they are attached to be detected, and/or furtherquantified if desired. Many appropriate detectable (imaging) agents areknown in the art, as are methods for their attachment to polypeptides.

Polypeptides are conjugated to a wide variety of fluorescent dyes,quenchers, and haptens such as fluorescein, R-phycoerythrin, and biotinin some embodiments. In some embodiments, conjugation occurs eitherduring polypeptide synthesis or after the polypeptide has beensynthesized and purified.

Alternatively, an antibody, antigen-binding fragment or binding proteinis conjugated with a fluorescent moiety in some embodiments. Conjugatingpolypeptides with fluorescent moieties (e.g., R-Phycoerythrin,fluorescein isothiocyanate (FITC), etc.) is, for example, accomplishedusing art-recognized techniques. Numerous commercially availablefluorescent dyes and dye-conjugation kits are commercially available forparticular applications, such as fluorescence microscopy, flowcytometry, fluorescence-activated cell sorting (FACS), etc.

In one non-limiting embodiment, an antibody antigen-binding fragment isassociated with (conjugated to) a detectable label, such as aradionuclide, a dye, an imaging agent, or a fluorescent agent forimmunodetection of binding to antigen which is used to visualize bindingof the antibodies to M2 macrophages or soluble or bound huCD163 proteinin vitro and/or in vivo.

Non-limiting examples of radiolabels include, for example, ³²P, ³³P,⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁴Cu, ⁶⁷Ga, ⁶⁷Cu, ⁶⁸Ga, ⁷¹Ge, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁷As,⁷⁷Br, ⁸¹Rb/^(81m)Kr, ^(87m)Sr, ⁹⁰Y, ⁹⁷Ru, ⁹⁹Tc, ^(99m)Tc, ¹⁰⁰Pd, ¹⁰¹Rh,¹⁰³Pb, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹Ag, ¹¹¹In, ¹¹³In, ¹¹⁹Sb, ¹²¹Sn, ¹²³I, ¹²⁵I,¹²⁷Cs, ¹²⁸Ba, ¹²⁹Cs, ¹³¹I, ¹³¹Cs, ¹⁴³Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁹Eu,¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹¹Os, ¹⁹³Pt, ¹⁹⁴Ir, ¹⁹⁷Hg, ¹⁹⁹Au, ²⁰³Pb,²¹¹At, ²¹²Pb, ²¹²Bi, and ²¹³Bi. In some embodiments, radiolabels areattached to compounds using conventional chemistry known in the art ofantibody imaging. Radiolabeled compounds are useful in in vitrodiagnostics techniques and in in vivo radioimaging techniques and inradioimmunotherapy.

Compositions of antibodies and antigen-binding fragments describedherein are also used as non-therapeutic agents (e.g., as affinitypurification agents) in some embodiments.

Antibody Technology

As will be understood by the skilled artisan, general description ofantibodies herein and methods of preparing and using the same also applyto individual antibody polypeptide constituents and antibody fragments.

The antibodies of the present disclosure are polyclonal or monoclonalantibodies. However, in preferred embodiments, they are monoclonal. Inparticular embodiments, antibodies of the present disclosure are humanantibodies. Methods of producing polyclonal and monoclonal antibodiesare known in the art.

Antibodies, antigen-binding fragments, and other proteins that bindhuCD163 expressed by M2 macrophages are generated using such methods aretested for one or more of their binding affinity, avidity, andmodulating capabilities in some embodiments.

Conventional methods, in some embodiments, are utilized to identifyantibodies or antigen-binding fragments thereof that bind to a huCD163protein. In some embodiments, antibodies and antigen-binding fragmentsare evaluated for one or more of binding affinity, association rates,disassociation rates, and avidity. Measurement of such parameters is,for example, accomplished using assays including, but not limited to, anenzyme-linked-immunosorbent assays (ELISA), ELISpot assays, Scatchardanalysis, surface plasmon resonance (e.g., BIACORE) analysis, etc.,competitive binding assays, and the like. In one non-limitingembodiment, an ELISA assay is used to measure the binding capability ofspecific antibodies or antigen-binding fragments that bind to a huCD163protein. A surface plasmon resonance technique is described in Liljebladet al., Glyco J 17:323-9 (2000).

In some embodiments, antibodies according to the disclosure are producedrecombinantly, using vectors and methods available in the art, asdescribed further below. In some embodiments, human antibodies are alsobe generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610and 5,229,275).

In some embodiments, human antibodies are produced in transgenic animals(e.g., mice) that are capable of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JO gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene arrayinto such germ-line mutant mice results in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., ProcNatl Acad Sci USA, 90:2551 (1993); Jakobovits et al., Nature 362:255-58(1993); Bruggemann et al., Year in Immunol., 7:33 (1993); U.S. Pat. Nos.5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97/17852. In someembodiments, such animals are genetically engineered to produce humanantibodies comprising a polypeptide of the present disclosure.

The antibodies are, for example, isolated and purified from a culturesupernatant or ascites (if produced in an animal) using methods known inthe art, such as by saturated ammonium sulfate precipitation, euglobulinprecipitation method, caproic acid method, caprylic acid method, ionexchange chromatography (DEAE or DE52), or affinity chromatography usinganti-Ig column or a protein A, G, or L column.

As noted above, the disclosure further provides antibody fragments. Incertain circumstances there are advantages of using antibody fragments,rather than whole antibodies. For example, the smaller size of thefragments allows for rapid clearance, and leads to improved access tocertain tissues, such as solid tumors, in some embodiments. Examples ofantibody fragments include: Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibodies; and multispecificantibodies formed from antibody fragments.

Various techniques have been developed to produce antibody fragments.Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies (see, e.g., Morimoto et al., J Biochem Biophys Methods1992 24(1-2):107-17; and Brennan et al., Science 1985 229:81). However,these fragments are now be produced directly by recombinant host cellsin some embodiments. In some embodiments, Fab, Fv, and ScFv antibodyfragments all are expressed in and secreted from E. coli, thus allowingthe facile production of large amounts of these fragments. In someembodiments, Fab′-SH fragments are directly recovered from E. coli andchemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach, insome embodiments, F(ab′)₂ fragments are isolated directly fromrecombinant host cell culture. Fab and F(ab′)₂ fragment with increasedin vivo half-life comprising a salvage receptor binding epitope takenfrom two loops of a C_(H)2 domain of an Fc region of an IgG aredescribed in U.S. Pat. Nos. 5,869,046 and 6,121,022. Other techniquesfor producing antibody fragments will be apparent to the skilledpractitioner.

In other embodiments, the antibody of choice is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions. Thus, they are suitable for reducednonspecific binding during in vivo use. In some embodiments, sFv fusionproteins are constructed to yield fusion of an effector protein ateither the amino or the carboxy terminus of an sFv. See AntibodyEngineering, ed. Borrebaeck, supra. In some embodiments, the antibodyfragment is also be a “linear antibody,” e.g., as described in U.S. Pat.No. 5,641,870 for example. In some embodiments, such linear antibodyfragments are monospecific or bispecific.

Methods for making bispecific or other multispecific antibodies areknown in the art and include chemical cross-linking, use of leucinezippers (Kostelny et al., J Immunol 148:1547-53 (1992)); diabodytechnology (Hollinger et al., Proc Natl Acad Sci USA 90:6444-8 (1993));scFv dimers [Gruber et al., J Immunol 152:5368 (1994)), linearantibodies (Zapata et al., Protein Eng 8:1057-62 (1995)); and chelatingrecombinant antibodies (Neri et al., J Mol Biol 246:367-73 (1995)).

Traditional production of full-length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature 305:537-9 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. Purification of thecorrect molecule are done, for example, by affinity chromatography.Similar procedures are disclosed in WO 93/08829, and in Traunecker etal., EMBO J 10:3655-9 (1991).

According to a different approach, antibody variable regions with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. Preferably, thefusion is with an Ig heavy chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. It is preferred that thefirst heavy-chain constant region (C_(H)1) containing the site necessaryfor light chain bonding, be present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host cell. This providesfor greater flexibility in adjusting the mutual proportions of the fourpolypeptide fragments in embodiments when unequal ratios of the fourpolypeptide chains used in the construction provide the optimum yield ofthe desired bispecific antibody. It is, however, possible to insert thecoding sequences for two or all four polypeptide chains into a singleexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios have nosignificant effect on the yield of the desired chain combination.

Bispecific antibodies are composed of, for example, a hybridimmunoglobulin heavy chain with a first binding specificity in one arm,and a hybrid immunoglobulin heavy chain-light chain pair (providing asecond binding specificity) in the other arm. This asymmetric structurefacilitates the separation of the desired bispecific compound fromunwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation. This approach is disclosed inWO 94/04690. For further details of generating bispecific antibodiessee, for example, Suresh et al., Methods Enzymol 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules are engineered tomaximize the percentage of heterodimers that are recovered fromrecombinant cell culture in some embodiments. The preferred interfacecomprises at least a part of the C_(H)3 domain. In this method, one ormore small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g., tyrosineor tryptophan). Compensatory “cavities” of identical or similar size tothe large side chain(s) are created on the interface of the secondantibody molecule by replacing large amino acid side chains with smallerones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugateare coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/20373, and EP 03089). In some embodiments,heteroconjugate antibodies are made using any convenient cross-linkingmethods. Suitable cross-linking agents are well known in the art, andare disclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques. Another method is designed to make tetramersby adding a streptavidin-coding sequence at the C-terminus of the scFv.Streptavidin is composed of four subunits, so when the scFv-streptavidinis folded, four subunits associate to form a tetramer (Kipriyanov etal., Hum Antibodies Hybridomas 6(3):93-101 (1995)).

According to another approach for making bispecific antibodies, theinterface between a pair of antibody molecules are engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture in some embodiments. One interface comprises atleast a part of the C_(H)3 domain of an antibody constant domain. Inthis method, one or more small amino acid side chains from the interfaceof the first antibody molecule are replaced with larger side chains(e.g., tyrosine or tryptophan). Compensatory “cavities” of identical orsimilar size to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chainswith smaller ones (e.g., alanine or threonine). This provides amechanism for increasing the yield of the heterodimer over otherunwanted end-products such as homodimers. See WO 96/27011.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies are prepared using chemical linkage. Brennan et al., Science229: 81 (1985) describes a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent, sodiumarsenite, to stabilize vicinal dithiols, and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. In some embodiments, the bispecificantibodies produced are used as agents for the selective immobilizationof enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which are, for example, chemically coupled to formbispecific antibodies. Shalaby et al., J Exp Med 175: 217-25 (1992)describes the production of a humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J Immunol 148(5):1547-53 (1992). Theleucine zipper peptides from the Fos and Jun proteins were linked to theFab′ portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. This method is be used toproduce antibody homodimers in some embodiments.

Identification and Preparation of Antibodies

Polynucleotide sequences encoding the antibodies, variable regionsthereof, or antigen-binding fragments thereof are, in some embodiments,determined using conventional sequencing techniques, and subcloned intoexpression vectors for the recombinant production of the antibodies.This was accomplished by obtaining mononuclear cells from the blood of asubject, e.g., a cancer patient; producing B cell clones from themononuclear cells; inducing the B cells to become antibody-producingplasma cells; and screening the supernatants produced by the plasmacells to determine if it contains an antibody. Identification of otherantibodies having the specificity of the antibodies of the disclosureare accomplished using a similar method in some embodiments. Forexample, once a B cell clone that produces an antibody is identified,reverse-transcription polymerase chain reaction (RT-PCR) is performed toclone the DNAs encoding the variable regions or portions thereof of theantibody. These sequences are then subcloned into expression vectorssuitable for the recombinant production of human antibodies. The bindingspecificity is confirmed, in some embodiments, by determining theantibody's or the recombinant antibody's ability to bind M2 cells orother cells expressing a human CD163 polypeptide that is expressed by M2cells.

In particular embodiments of the methods described herein, B cellsisolated from peripheral blood or lymph nodes are sorted, e.g., based ontheir being CD19 positive, and plated, e.g., as low as a single cellspecificity per well, e.g., in 96-, 384-, or 1536-well configurations.The cells are induced to differentiate into antibody-producing cells,e.g., plasma cells, and the culture supernatants are harvested andtested for binding to cells expressing the target polypeptide on theirsurface using, e.g., FMAT or FACS analysis. Positive wells are thensubjected to whole well RT-PCR to amplify heavy and light chain variableregions of the IgG molecule expressed by the clonal daughter plasmacells. The resulting PCR products encoding the heavy and light chainvariable regions, or portions thereof, are subcloned into human antibodyexpression vectors for recombinant expression. The resulting recombinantantibodies are then tested to confirm their original binding specificityand are further tested, in some embodiments, for cross-reactivityagainst other cells or proteins.

Thus, in one embodiment, a method of identifying antibodies is practicedas follows. First, full-length or approximately full-length CD163 cDNAsare transfected into a cell line for expression of CD163 polypeptides.Secondly, individual human plasma or sera samples are tested forantibodies that bind the cell-expressed polypeptides. And lastly, MAbsderived from plasma- or serum-positive individuals are characterized forbinding to the same cell-expressed CD163 polypeptides. Furtherdefinition of the fine specificities of the MAbs are performed at thispoint in some embodiments.

Polynucleotides that encode the antibodies or portions thereof of thepresent disclosure are isolated from cells expressing the antibodies,according to methods available in the art and described herein,including amplification by polymerase chain reaction using primersspecific for conserved regions of human antibody polypeptides, in someembodiments. For example, light chain and heavy chain variable regionsis cloned from the B cell according to molecular biology techniquesdescribed in WO 92/02551; U.S. Pat. No. 5,627,052; or Babcook et al.,Proc Natl Acad Sci USA 93:7843-48 (1996). In certain embodiments,polynucleotides encoding all or a region of both the heavy and lightchain variable regions of the IgG molecule expressed by the clonaldaughter plasma cells expressing the antibody are subcloned andsequenced. In some embodiments, the sequence of the encoded polypeptideis readily determined from the polynucleotide sequence.

Isolated polynucleotides encoding a polypeptide of the presentdisclosure is subcloned into an expression vector to recombinantlyproduce antibodies and polypeptides of the present disclosure, usingprocedures known in the art and described herein.

In some embodiments, binding properties of an antibody (or fragmentthereof) to CD163 polypeptides or M2 cells are generally determined andassessed using immunodetection methods including, for example,immunofluorescence-based assays, such as immuno-histochemistry (IHC)and/or fluorescence-activated cell sorting (FACS). Immunoassay methodsinclude, in some embodiments, controls and procedures to determinewhether antibodies bind specifically to CD163 polypeptides or to M2macrophages, and do not recognize or cross-react with control cells,e.g., M1 cells, or host cells transfected to express a control protein.

Following pre-screening of serum to identify patients that produceantibodies to a CD163 polypeptide or to M2 macrophages or TAMs, themethods of the present disclosure typically include the isolation orpurification of B cells from a biological sample previously obtainedfrom a patient or subject. In some embodiments, the patient or subjectare currently or have previously been diagnosed with or suspect orhaving a cancer or particular disease of interest, or the patient orsubject is considered free of cancer or disease. Typically, the patientor subject is a mammal and, in particular embodiments, a human. In someembodiments, the biological sample is any sample that contains B cells,including but not limited to, lymph node or lymph node tissue, pleuraleffusions, peripheral blood, ascites, tumor tissue, or cerebrospinalfluid (CSF). In some embodiments, B cells are isolated from differenttypes of biological samples, such as a tumor biopsy or other biologicalsample affected by a particular disease. However, in some embodiments,it is understood that any biological sample comprising B cells is usedfor any of the embodiments of the present disclosure.

Once isolated, the B cells are induced to produce antibodies, e.g., byculturing the B cells under conditions that support B cell proliferationor development into a plasmacyte, plasmablast, or plasma cell. Theantibodies are then screened, typically using high throughputtechniques, to identify an antibody that specifically binds to a targetantigen, e.g., a particular tissue, cell, or polypeptide. In certainembodiments, the specific antigen, e.g., cell surface polypeptide boundby the antibody is not known, while in other embodiments, the antigenspecifically bound by the antibody is known.

According to the present disclosure, B cells are, in some embodiments,isolated from a biological sample, e.g., a tumor, tissue, peripheralblood or lymph node sample, by any means known and available in the art.B cells are typically sorted by FACS based on the presence on theirsurface of a B cell-specific marker, e.g., CD19, CD138, and/or surfaceIgG. However, other methods known in the art are employed in someembodiments, such as, e.g., column purification using CD19 magneticbeads or IgG-specific magnetic beads, followed by elution from thecolumn. However, magnetic isolation of B cells utilizing any markerresults in loss of certain B cells in some embodiments. Therefore, incertain embodiments, the isolated cells are not sorted but, instead,Ficoll-purified mononuclear cells isolated from tumor are directlyplated to the appropriate or desired number of specificities per well.

To identify B cells that produce an antibody, the B cells are typicallyplated at low density (e.g., a single cell specificity per well, 1-10cells per well, 10-100 cells per well, 1-100 cells per well, less than10 cells per well, or less than 100 cells per well) in multi-well ormicrotiter plates, e.g., in 96, 384, or 1536 well configurations. Whenthe B cells are initially plated at a density greater than one cell perwell, then the methods of the present disclosure include the step ofsubsequently diluting cells in a well identified as producing anantigen-specific antibody, until a single cell specificity per well isachieved, thereby facilitating the identification of the B cell thatproduces the antigen-specific antibody in some embodiments. In someembodiments, cell supernatants or a portion thereof and/or cells arefrozen and stored for future testing and later recovery of antibodypolynucleotides.

In certain embodiments, the B cells are cultured under conditions thatfavor the production of antibodies by the B cells. For example, the Bcells are cultured under conditions favorable for B cell proliferationand differentiation to yield antibody-producing plasmablasts,plasmacytes, or plasma cells. In particular embodiments, the B cells arecultured in the presence of a B cell mitogen, such as lipopolysaccharide(LPS) or CD40 ligand. In one specific embodiment, B cells aredifferentiated to antibody-producing cells by culturing them with feedcells and/or other B cell activators, such as CD40 ligand.

Cell culture supernatants or antibodies obtained therefrom are testedfor their ability to bind to a target antigen, using routine methodsavailable in the art, including those described herein, in someembodiments. In particular embodiments, culture supernatants are testedfor the presence of antibodies that bind to a target antigen usinghigh-throughput methods. For example, B cells are cultured in multi-wellmicrotiter dishes, such that robotic plate handlers are used tosimultaneously sample multiple cell supernatants and test for thepresence of antibodies that bind to a target antigen. In particularembodiments, antigens are bound to beads, e.g., paramagnetic or latexbeads) to facilitate the capture of antibody/antigen complexes. In otherembodiments, antigens and antibodies are fluorescently labeled (withdifferent labels) and FACS analysis is performed to identify thepresence of antibodies that bind to target antigen. In one embodiment,antibody binding is determined using FMAT™ analysis and instrumentation(Applied Biosystems, Foster City, Calif.). FMAT is a fluorescencemacro-confocal platform for high-throughput screening, which enablesmix-and-read, non-radioactive assays using live cells or beads.

In comparing the binding of an antibody to a particular target antigen(e.g., a biological sample such as cancer tissue or cells, or infectiousagents) to the antibody's binding to a control sample (e.g., abiological sample such as normal cells, comparator cells from anotherspecies, a different cancer tissue or cell, or different infectiousagent), in some embodiments, the antibody is considered topreferentially bind a particular target antigen if at least two-fold, atleast three-fold, at least five-fold, or at least ten-fold more antibodybinds to the particular target antigen as compared to the amount thatbinds a control sample.

Polynucleotides encoding antibody chains, variable regions thereof, orfragments thereof, are isolated from cells utilizing any means availablein the art in some embodiments. In one embodiment, polynucleotides areisolated using polymerase chain reaction (PCR), e.g., reversetranscription-PCR (RT-PCR) using oligonucleotide primers thatspecifically bind to heavy or light chain encoding polynucleotidesequences or complements thereof using routine procedures available inthe art. In one embodiment, positive wells are subjected to whole wellRT-PCR to amplify the heavy and light chain variable regions of the IgGmolecule expressed by the clonal daughter plasma cells. These PCRproducts, in some embodiments, are sequenced, and products encoding theheavy and light chain variable regions or portions thereof are thensubcloned into human antibody expression vectors and recombinantlyexpressed according to routine procedures in the art (see, e.g., U.S.Pat. No. 7,112,439). The nucleic acid molecules encoding a M2macrophage-specific antibody or fragment thereof as described hereinare, in some embodiments, propagated and expressed according to any of avariety of well-known procedures for nucleic acid excision, ligation,transformation, and transfection. Thus, in certain embodimentsexpression of an antibody fragment are preferred in a prokaryotic hostcell, such as E. coli (see, e.g., Pluckthun et al., Methods Enzymol178:497-515 (1989)). In certain other embodiments, expression of theantibody or an antigen-binding fragment thereof are preferred in aeukaryotic host cell, such as yeast (e.g., Saccharomyces cerevisiae, S.pombe, Pichia pastoris); animal cells (including mammalian cells); orplant cells. Examples of suitable animal cells include, but are notlimited to, myeloma, COS, CHO, or hybridoma cells. Examples of plantcells include tobacco, corn, soybean, and rice cells. By methods knownto those having ordinary skill in the art and based on the presentdisclosure, a nucleic acid vector is designed for expressing foreignsequences in a particular host system, and then polynucleotide sequencesencoding the tumor-specific antibody (or fragment thereof) is inserted,in some embodiments. The regulatory elements will vary according to theparticular host.

One or more replicable expression vectors containing a polynucleotideencoding a variable and/or constant region is, in some embodiments,prepared and used to transform an appropriate cell line, for example, anon-producing myeloma cell line, such as a mouse NSO line or abacterium, such as E. coli, in which production of the antibody willoccur. In order to obtain efficient transcription and translation, thepolynucleotide sequence in each vector should include appropriateregulatory sequences, particularly a promoter and leader sequenceoperatively linked to the variable region sequence.

Particular methods for producing antibodies in this way are generallywell known and routinely used. For example, molecular biology proceduresare described by Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989; see alsoSambrook et al., 3rd ed., Cold Spring Harbor Laboratory, New York,(2001)). While not required, in certain embodiments, regions ofpolynucleotides encoding the recombinant antibodies are sequenced. DNAsequencing are performed, for example, in any manner or using anysystems known in the art. Basic sequencing technology is described forexample, in Sanger et al., Proc Natl Acad Sci USA 74:5463 (1977)) andthe Amersham International plc sequencing handbook and includingimprovements thereto.

In particular embodiments, the resulting recombinant antibodies orfragments thereof are then tested to confirm their original specificity,and are further tested for cross-reactivity, e.g., with relatedpolypeptides, in some embodiments. In particular embodiments, anantibody identified or produced according to methods described herein istested for ability to internalize or other effector function usingconventional methods.

Packages, Kits, and Pre-Filled Containers

Also provided herein are kits containing one or more compounds describedabove. The kit comprises, in some embodiments, an antibody orantigen-binding fragment thereof as described herein in suitablecontainer means.

In some embodiments, there is provided is a container means comprising acomposition described herein. In some embodiments, the container meansis any suitable container which houses, for example, a liquid orlyophilized composition including, but not limited to, a vial, syringe,bottle, an in intravenous (IV) bag or ampoule. A syringe holds anyvolume of liquid suitable for injection into a subject, in someembodiments, including, but not limited to, 0.5 cc, 1 cc, 2 cc, 5 cc, 10cc, or more.

Provided herein are kits, comprising a composition or compositionsdescribed herein. In some embodiments, provided herein is a kit fortreating a subject having a cancer, comprising an antibody as describedherein and an anticancer therapy.

In some embodiments, provided herein is a kit for treating a cancer,comprising an antibody as described herein, and a label attached to orpackaged with the container, the label describing use of the antibody incombination with an anticancer therapy.

In some embodiments, provided herein is a kit for treating a cancer,comprising an anticancer therapy and a label attached to or packagedwith the container, the label describing use of the anticancer therapywith an antibody as described herein.

In some embodiments, the container means of the kits will generallyinclude at least one vial, test tube, flask, bottle, ampoule, syringe anintravenous (IV) bag, and/or other container means, into which the atleast one polypeptide are placed, and/or preferably, suitably aliquoted.Provided herein is a container means comprising a composition describedherein.

The kits, in some embodiments, include a means for containing at leastone fusion protein, detectable moiety, reporter molecule, and/or anyother reagent containers in close confinement for commercial sale. Insome embodiments, such containers include injection and/or blow-moldedplastic containers into which the desired vials are retained. In someembodiments, kits also include printed material for use of the materialsin the kit.

Packages and kits additionally include a buffering agent, apreservative, and/or a stabilizing agent in a pharmaceutical formulationin some embodiments. In some embodiments, each component of the kit isenclosed within an individual container and all of the variouscontainers can be within a single package. In some embodiments,disclosure kits are designed for cold storage or room temperaturestorage.

Additionally, in some embodiments, the preparations contain stabilizersto increase the shelf-life of the kits and include, for example, bovineserum albumin (BSA). Where the compositions are lyophilized, the kitcontains, in some embodiments, further preparations of solutions toreconstitute the lyophilized preparations. Acceptable reconstitutionsolutions are well known in the art and include, for example,pharmaceutically acceptable phosphate buffered saline (PBS).

In some embodiments, packages and kits further include one or morecomponents for an assay, such as, for example, an ELISA assay. Samplesto be tested in this application include, for example, blood, plasma,tissue sections and secretions, urine, lymph, and products thereof. Insome embodiments, packages and kits further include one or morecomponents for collection of a sample (e.g., a syringe, a cup, a swab,etc.).

In some embodiments, packages and kits further include a labelspecifying information required by US FDA or similar regulatoryauthority, for example, a product description, amount and mode ofadministration, and/or indication of treatment. Packages provided hereincan include any of the compositions as described herein.

The term “packaging material” refers to a physical structure housing thecomponents of the kit. In some embodiments, the packaging materialmaintains the components sterilely and are made of material commonlyused for such purposes (e.g., paper, corrugated fiber, glass, plastic,foil, ampules, etc.). In some embodiments, the label or packaging insertincludes appropriate written instructions. Kits, therefore, additionallyincludes, in some embodiments, labels or instructions for using the kitcomponents in any method of the disclosure. In some embodiments, a kitincludes a compound in a pack, or dispenser together with instructionsfor administering the compound in a method described herein.

In still further embodiments, a kit further comprises a container meansfor an anticancer therapy.

Instructions include instructions for practicing any of the methodsdescribed herein including treatment methods in some embodiments.Instructions additionally include indications of a satisfactory clinicalendpoint or any adverse symptoms that occur, or additional informationrequired by regulatory agencies such as the Food and Drug Administrationfor use on a human subject in some embodiments.

The instructions are, in some embodiments, on “printed matter,” e.g., onpaper or cardboard within or affixed to the kit, or on a label affixedto the kit or packaging material, or attached to a vial or tubecontaining a component of the kit. Instructions are additionallyincluded on a computer readable medium, such as, for example, CD-ROMs,DVDs, flash memory devices, solid state memory, magnetic disks and diskdevices, magnetic tapes, cloud computing systems and services, and thelike, in some embodiments. In some cases, the program and instructionsare permanently, substantially permanently, semi-permanently, ornon-transitorily encoded on the media.

Provided herein is a container means comprising a composition describedherein. In some embodiments, the container means is any suitablecontainer which houses a liquid or lyophilized composition including,but not limited to, a vial, syringe, bottle, intravenous (IV) bag, orampoule. A syringe, in some embodiments, holds any volume of liquidsuitable for injection into a subject including, but not limited to, 0.5cc, 1 cc, 2 cc, 5 cc, 10 cc or more.

Provided herein are kits comprising a composition described herein. Insome embodiments, provided herein is a kit for treating a cancer,comprising an antibody as described herein in combination with ananticancer therapy agent.

In some embodiments, provided herein is a kit for treating a cancer,comprising an antibody as described herein, and a label attached to orpackaged with the container, the label describing use of the antibody,or an antigen-binding fragment thereof, with an anticancer therapy.

In some embodiments, provided herein is a kit for treating a cancer,comprising an anticancer therapy and a label attached to or packagedwith the container, the label describing use of the anticancer therapywith an antibody as described herein.

EXAMPLES

The present disclosure will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the disclosure in any way.

Example 1—Identification and Cloning

Antibodies that specifically bind to human myeloid-derived suppressorcells (MDSCs) produced by patients who respond to checkpoint inhibitoranti-PD-1 treatment were isolated and cloned. These monoclonalantibodies were further interrogated for their immunomodulatoryproperties with the goal of identifying antibodies that have therapeuticpotential to target and reverse the immunosuppressive effects of MDSCs,thereby enhancing tumor clearance.

Cancer patients who achieved partial or complete response to immunecheckpoint inhibitor for at least 6 months of duration were identifiedand selected for memory B cell repertoire analysis via the I-STARplatform. This platform utilized a short-term B cell culture system tointerrogate the memory B cell repertoire. More than 15,000 memory Bcells based on CD19 and IgG surface-expression were isolated from tenmillion peripheral blood mononuclear cells (PBMCs) of each donorpatient. These memory B cells were then seeded in forty 384-wellmicrotiter plates, at approximately 1 cell/well, under conditions thatpromoted B cell activation, proliferation, terminal differentiation, andantibody secretion. The plating density of 1 cell/well allowed forexpansion of single B cell clones such that the authentic antibody heavyand light chain pair could be reconstituted from each culture well.Using a high throughput and miniaturized, multiplex flow cytometryassay, the secreted IgG antibodies in each well were screened forbinding to MDSCs. 49 positive B cell clones were identified. A selectedsubset of antibodies, prioritized based on MDSCs binding profiles andantibody variable-region sequences, was sequenced, cloned, and expressedas recombinant IgG1 for further in vitro characterizations.

Heavy (VH) and light (VL) variable regions of the immunoglobulin genesfrom B cell clones that produce MDSC-specific antibodies were amplifiedby RT-PCR amplification using family-specific primer sets. From positivefamily-specific PCR reactions, pools of the VH- or VL-region clones werecloned into an expression vector upstream to human IgG1 constant domainsequence, resulting in a functional antibody with the same bindingcharacteristic as the antibody produced by that B cell clone. DNAplasmids were designed and requested for gene synthesis in constantregions at GenScript, NJ, USA. These plasmids were combined in allpossible heavy and light chain family-specific pairs and were used totransiently transfect HEK293 cells. All transfectant supernatantscontaining secreted recombinant antibodies were screened in flow-basedMDSC binding assays. For wells that contained more than one B cell cloneper well, multiple VH and VL domain sequences were amplified andexpressed as described earlier. A MDSC screen was then used to identifythe heavy and light chain combination pools that recapitulated thebinding activity as observed with the antibody found in the mixedcultures. DNA sequences of the VH and light VL variable regions for allbinding mAbs were confirmed by multiple sequencing reactions usingpurified DNA from maxipreps (GenScript source and Macherey Nagelamplified plasmids).

One B cell clone (Germline ID for heavy chain VH3.30-3/IGHG1 and lightchain VK1.O12) was identified from MDSC screen and designated AB101comprising a light chain comprising SEQ ID NO: 9 and a heavy chaincomprising SEQ ID NO: 10. The donor from whom the clone was derived wasa patient diagnosed with non-small-cell lung cancer (NSCLC). The patienthad progressive disease to chemotherapy and then had complete remissionupon anti-PD-1 treatment and was still receiving treatment at the timeof blood draw. The B cell clone well was confirmed to have just oneheavy and one light chain from sequencing. As observed with the secretedIgG antibodies from the single B cell clone, the recapitulated antibodyalso had a distinct bimodal binding on MDSCs, indicating that theantibody target is highly expressed on a select subpopulation of MDSCs.See FIG. 1 . Results from recapitulation screen on two MDSC donors withrelaxed block (10 μg/mL of recombinant Fc block (Abcam) and stringentblock (10 μg/mL of recombinant Fc block from Abcam+1 μg/mL of anti-CD16,anti-CD32, and anti-CD64) conditions show dose-dependent saturablebinding of AB101 to human MDSCs with an IC50 of about 10 nM underrelaxed block conditions. There was a decrease in overall binding ofAB101, as suggested by decrease in MFI, under stringent blockconditions.

CD163 is a marker of cells from the monocyte/macrophage lineage. Theexpression of CD163 on in vitro differentiated MDSCs has been reportedto be bimodal. It was hypothesized that bimodal binding of AB101 maycorrelate with CD163 expression. To test this hypothesis, in vitro MDSCswere generated (see EXAMPLE 11) and co-stained with anti-CD163(BioLegend 333611) and with AF647 conjugated AB101 as described inEXAMPLE 8 and analyzed for binding by FACS. Cells were first gated asCD163 high or low and then examined for binding with variousconcentrations of AB101 or human IgG1 isotype control. The subpopulationof cells that the AB101 antibody was binding to was CD163^(Hi) cells.See FIG. 2 .

Example 2—Isolation of Autologous Monocytes and T Cells

This example shows the isolation of autologous monocytes and T cells.Human monocytes and T cells were obtained using techniques commonly usedin the art. Human monocytes and T cells were isolated from white bloodcells (WBCs) trapped within an integrated chamber, known as theLeukoReduction System (LRS) chambers, during the plateletpheresiscollection process. Peripheral blood mononuclear cells (PBMC) werepurified from the LRS samples by standard density gradientcentrifugation (Ficoll™ Paque Premium 1.073, GE Healthcare No.17-5449-52). The supernatant was discarded, and the pellet resuspendedin 20 mL EasySep™ Buffer (StemCell Technologies No. 20144) forenumeration of PBMCs and further isolations of monocytes and T cells.

Monocytes were isolated using the EasySep Human Monocyte Isolation kit(Stem Cell No. 19359) following the manufacturer's instructions.

Total CD3, CD4, or CD8 T cells were isolated using the Human CD3⁺ T CellIsolation Kit (StemCell 19051), EasySep™ Human CD4⁺ T Cell Isolation Kit(StemCell No. 17952), EasySep™ Human CD8⁺ T Cell Isolation Kit (StemCellNo. 17953), respectively, following the manufactures instructions. Thesenegative selection kits used antibodies to label undesired cell typesfor removal, allowing the desired target cells to be isolated from thesample.

Example 3—AB101 Specific Binding to Immunosuppressive Myeloid Cells

To assess specificity, the binding of an antibody of the invention,antibody AB101, conjugated to the far-red fluorescence dye AF647, wastested on different cell types, including MDSCs, immune suppressive M2cand pro-inflammatory M1 macrophages generated as described in EXAMPLE 11and below. Additionally, separate immune populations from PBMCs ofhealthy donors were assessed for antibody binding. For each of thesestudies at least 3 individual donors were used. PBMCs were isolated fromblood using a Ficoll gradient using standard procedures. To distinguishthe immune cell populations, PBMC cells were then stained withhematopoietic lineage markers (CD45-BV421 (BD 642275). CD3-BV510 (BD563109), CD11c-PE-Cy7 (BD 561356), CD14-FITC (BD 347493), CD20-APC-Cy7(BD 562643), CD56-PE (BD 347747), and CD66c (BD 551478). Separatelineage populations were further characterized by the followingexpression patterns and assessed for binding to AB101: T cellsCD45⁺CD3⁺; B cells CD45⁺CD20⁺; monocytes CD45⁺CD14⁺, NK cellsCD45⁺SSC^(low)CD14⁻CD3⁻CD56⁺; granulocytes CD45⁺SSC^(Hi)CD14⁻CD66⁺; anddendritic cells CD45⁺CD14⁻CD66⁻CD11c⁺. Antibody binding to primary humannon-immune cells (Lonza) was also assessed, including small airwayepithelial cells (SAEC, #CC-2547), renal proximal tubule epithelialcells (RPTEC, #CC-2553), lung microvascular endothelial cells (HMVEC,#CC-2527), umbilical vein endothelial cells (HUVEC, #C2519A), aorticsmooth muscle cells (AOSMC, #CC2571), and keratinocytes (#00192627).These cells were cultured in cell type-specific medium and conditionsper manufacturer's instructions until 60-70% confluency, then thenharvested to test for antibody binding by flow cytometry (FIG. 4 andFIG. 5 ).

In vitro monocytic MDSCs were generated from isolated monocytes bystandard methods: Day 0, monocytes were plated in RPMI 1640 (HycloneSH30027.02, serum-free) at 1.5×10⁵/cm², incubated for 1 hour at 5% CO₂and 37° C., then washed with pre-warmed RPMI before adding MDSC medium(RMPI+10% FBS (Hyclone SH30070.03)+50 ng/mL GM-CSF (R&D Systems215-GM-010)+50 ng/mL IL-6 (R&D Systems 206-IL-010), 20 mL per T75 flask)to the cells. Cells were then cultured in 5% CO₂, 37° C. for 7 dayswithout medium change. After 7 days, cells were harvested by washing 2×with PBS (Hyclone SH30028.03)+2 mM EDTA then adding cold MacrophageDetachment Solution (PromoCell C-41330) at 15 mL per T75 flask followedby incubation for 40 min at 2-8° C. Cells were dislodged by tapping theflask against the palm, collected and diluted 1:1 with PBS+2 mM EDTA.Cells were pelleted in a conical tube by centrifuging for 15 min at450×g, washed once with PBS+2 mM EDTA, counted and resuspended at 1×10⁷per mL in FACS blocking buffer (PBS+1% FBS+0.1 μg/mL Fc block (AbcamAb90285) for relaxed staining conditions, or PBS+1% FBS+Fc block and+0.01 μg/mL CDR block (antibodies against FcR CD16, CD32, and CD64; BDBiosciences 556617, 557333, and 555525 respectively) for stringentstaining conditions). Cells were incubated in FACS blocking buffer for20 minutes (min) at room temperature (RT) then 30 min at 4° C. The cellswere then diluted to 1×10⁶ cells/mL with FACS buffer+5% BSA (SigmaA3059) and 40 μL of cells (4×10⁴ cells) were aliquoted to wells forstaining. Primary antibodies (AB101 at 20, 6, 2, 0.75, 0.25, 0.08, and0.02 μg/mL or Human IgG1 Isotype control at 6 μg/mL) were added to thecells and incubated for 90 min at RT. The cells were washed 3× with 250μL/well of FACS buffer+5% BSA. Secondary APC Goat anti-Human IgG(Jackson IR 109-136-097) antibodies were prepared at 1:250 in FACSbuffer+BSA+e780 viability dye (at 1:1000 dilution) and added to cells(50 μL per well). After incubation at 4° C. for 45 minutes, cells werewashed 3× in 250 μL FACS buffer. Cells were then fixed in 100 μL perwell of 4% PFA for 10-15 min at RT, washed once with 250 μL FACS buffer,pelleted at 650×g for 5 min, then resuspended in 100 μL of FACS bufferfor analysis by flow cytometry.

The AB101 antibody binds to human immunosuppressive myeloid cells (M2cmacrophages and monocytic MDSCs) as show in FIG. 3 , which plots the MFIof AB101 or isotype staining on M2c, M1, and M0. The AB101 antibody doesnot bind to B, T and NK cells, and granulocytes as illustrated in FIG. 4, which shows staining of AB101 on T, B and NKT cells, neutrophils,monocytes, and dendritic cells (black curve) compared to isotype control(gray curve), and non-immune cells such as SAEC, RPTEC, HMVEC, HUVEC,AOSMC, and keratinocytes as shown in FIG. 5 . Thus, the AB101 antibodyspecifically binds to CD163-expressing immune-suppressive myeloid cellswithout impacting other immune or non-immune cells.

Example 4—AB101 FcNull Antibody Immunoprecipitates CD163 Polypeptide

This example shows immunoprecipitation of CD163 using an FcNull antibodycomprising AB101 variable domains in an IgG1 sequence modified tosubstantially reduce binding of the antibody to Fc receptors (Fc null),designated AB102 comprising a light chain comprising SEQ ID NO: 9 and aheavy chain comprising SEQ ID NO: 11. Immunoprecipitation (IP) wasperformed based on Klockenbusch and Kast, J Biomed Biotechnol Article ID927585 (2010). Antibodies were added prior to paraformaldehyde (PFA)fixative in a more classic cross-linked IP approach or prior tobis(sulfocuccinimidyl)suberate (BS3) crosslinking followed by an IP.

For IP involving the cross-linking using PFA approach, monocytes wereisolated from human blood and then polarized into M2 cells using theprotocol of EXAMPLE 2 and EXAMPLE 11, M2 macrophages were detached fromthe plate after incubation with macrophage detachment solution(Macrophage Detachment Solution DXF; PromoCell, No. C-41330) at 37° C.for ˜10 min, during which cells were rounded up and beginning to detach.The flasks were firmly tapped to facilitate cell detachment. Afterdetachment the macrophage detachment solution was quenched by additionof FACS buffer to the cells. The cells were pelleted at 300×g for 10 minand the supernatant was removed. The cell pellet was resuspended in 30mL of PBS containing 5% BSA (w/v) and 1 mM EDTA pH 8.0 and wereincubated on ice for 30 min.

The cells were split into 6 aliquots of 15×10⁶ cells per aliquot (5 mLper aliquot) and biotinylated antibodies were added to each. Twoaliquots received 50 μg each of the mouse IgG1 anti-hCD163 (R&D, No.MAB1607), two aliquots received 100 μg each of an isotype-controlantibody ISO2 (in Fc null framework) and two aliquots received 100 μgeach of test antibody AB102 (comprising AB101 variable domains in anIgG1 sequence modified to substantially reduce binding of the antibodyto Fc receptors (Fc null)). Cells were incubated at 4° C. for 1 hour(hr), with occasional gentle mixing by inversion of the tubes. Cellswere pelleted at 300×g for 5 min and washed 3× with PBS-EDTA and thenresuspended in 5 mL PBS (without magnesium or calcium; HyClone, No.SH30028.02). Paraformaldehyde (PFA; 5 mL of 0.8%) was added to eachtube, for a final PFA concentration of 0.4%. The cells were incubated inPFA at room temp for 5 min with gentle rocking. The cells were pelletedby centrifugation at 800×g for 5 min and the supernatant was removed.The cells were resuspended in 10 mL of ice-cold PBS containing 1.25 Mglycine to quench. The cells were pelleted at 800×g for 5 min andresuspended in ice-cold PBS. The cells were pelleted at 800×g for 5 minand resuspended in 1.0 mL of RIPA buffer containing 1× proteaseinhibitors (ThermoFisher Scientific, No. 89900). The cells wereincubated on ice for 2 hr and then passed through a 2 mL Douncehomogenizer 15 times.

Cell lysates were spun in a hanging bucket centrifuge to pellet nucleiand supernatants were used for the IPs. Protein lysate (50 μL) was setaside as the input fraction and 2.0 mL of cold PBS containing 1×protease inhibitors was added to the remaining supernatant. DynabeadsMyOne Streptavidin (250 μL) (ThermoFisher Scientific, No. 65601) wasadded to each sample and they were rotated overnight at 4° C. The nextday beads were collected with StemCell magnets and the supernatant wasremoved. The beads were sequentially washed with 5 mL of Paro Buffer I,5 mL of Paro Buffer II, and 5 mL Paro Buffer III for 5 min at 4° C.(Oncotarget. 2017; 8(7): 11105-11113). The beads were then washed 3×with cold PBS and finally resuspended in 100 μL, PBS and frozen at −80°C.

The beads were analyzed by mass spectrometry. This analysis wasperformed generally in accordance with the method described by Yan etal., Mol Cell Proteomics 10(3):M110.005611 (2011). In this method, thereversal of formaldehyde crosslinking and the elution of proteins fromstreptavidin beads was carried out the with 6 M guanidine, 150 mM Trisbuffer (pH 8.3) at 60° C. for 3 hr with constant agitation. Thesupernatant was denatured, reduced, and alkylated in the same bufferwith 10 mM tris(2-carboxyethyl)-phosphine (TCEP) and 50 mMchloroacetamide (CAA) at 95° C. for 10 min. The samples were thendiluted 10× and digested with 1.3 μg trypsin each overnight at 37° C.The peptides were cleaned by C18 cartridges. One replicate of AB102 IPand one replicate of anti-CD163 IP were eluted from C18 cartridges fordirect MS/MS analysis. One replicate of AB102 IP and one replicate ofanti-CD163 were incubated with 0.1 M formaldehyde-d2, 0.4 M sodiumcyanoborohydride in PBS buffer pH 7.5 for one hour to label with heavydimethylation (d4) and both replicates of ISO2 (Fc null) IPs wereincubated with 0.1 M formaldehyde, 0.4 M sodium cyanoborohydride in PBSbuffer pH 7.5 for one hour to label with light dimethylation (d0) on C18cartridges. Then the C18 cartridges were washed with 0.1%trifluoroacetic acid (TFA) and eluted by 80% acetonitrile (ACN). Thed0/d4 dimethylated peptides were resuspended in Buffer A (20% ACN, 0.1%TFA) and then mixed. The peptides were fractionated using an in-houseprepared microcapillary HPLC strong cation exchange column (SCX) (200mm×20 cm; Proteomix SCX 3 μm, Sepax Technologies). Peptides were loadedonto the microcapillary column equilibrated in Buffer A and washed withBuffer A. Bound peptides were eluted with 20 μL of Buffer A containing30%, 50% of Buffer B (800 mM ammonium formate, 20% ACN, pH 2.8),followed by 20 μL elution with Buffer D (0.5 M ammonium acetate, 50%ACN). All the samples were dried by Speed-Vac and directly analyzed byThermo-Oritrap-Fusion. The spectra were searched against human UniProtdatabase by Comet search engine(https://sourceforge.net/projects/comet-ms/). For demethylation labels,differential modification of 28.03 Da (for d0 dimethylation) and 32.06Da (for d4 dimethylation) on the N-termini and Lys sidechain were used.

The heavy/light samples were run through an in-line HPLC column usingstrong cation exchange column (200 mm×20 cm; Proteomix SCX 3 μm, SepaxTechnologies) and subjected to MS/MS using Thermo-Oritrap-Fusion. Forheavy and light labeled samples only spectra that were fully methylatedwere included. Eighty-nine proteins were identified where all peptidescontained the heavy isotope, indicating that the protein was identifiedin the AB102 IP but not in the negative control. Of the 89 proteinsunique to the AB102 IP, 12 were considered to be plausibly on the cellsurface: CD163, RIPK1, NEUA, SLC31, LRP8, SLIT1, RAF1, ILK, ATRN1,MCA32, FNBP2, and LRRN3. One additional protein, TNRS, was found to havetwo heavy methylated peptides and one unmethylated peptide (IP originunknown), suggesting it could also be exclusive to the AB102 IP.

Peptides from one replicate of AB102 IP and one replicate of the controlanti-CD163 IP were individually analyzed by mass spectrometry. Of the360 proteins identified in the AB102 IP, 45 of them were curated aspotentially membrane-bound or secreted. Of the proteins identified inother data sets, the following were found in the AB102 IP: CD163,Galectin-1, Galectin-3, and Peptidyl-Prolyl Cis-Trans Isomerase A(PPIA). Casein kinase IIb, which has been reported to interact directlywith CD163, was also identified in this dataset.

For IP by cross-linking using BS3 approach, macrophages were harvestedby collecting supernatants from flasks into 250 mL centrifuge tubes.Cold Macrophage Detachment Solution (30 mL) was added to each flask andincubated for 45 min at 4° C. The flasks were then scraped with ascraper and cells were collected into 250 mL centrifuge tubes andcentrifuged at 650×g for 10 min. The medium was aspirated, leaving thecell pellet in the tube. The cells were then resuspended in 20 mL coldPBS+2 mM EDTA.

The cells were then diluted to 1×10⁷ cells/mL with PBS+2 mM EDTA andwere split into three volumes of 40%, 40%, and 20% total volume. AB102(2.5 mg) was added to one of the 40% fractions. An anti-PDL1 antibody inan FcNull framework (2.5 mg) was added to the other 40% fraction, whichwas the positive control. The isotype control (ISO2 in FcNull framework,1.25 mg) was added to the 20% fraction, which was the negative control.Each fraction was incubated with gentle mixing for 2 hr at 4° C. Theywere then centrifuged at 650×g for 10 min and the supernatant wascarefully decanted. The pellets were each resuspended in 15 mL PBS+EDTAand then centrifuged at 650×g for 10 min. This wash step was thenrepeated. The wash buffer was next carefully removed without disturbingthe pellets. The pellets from the 40% fractions were resuspended in 2 mLcross-linking buffer. The pellet from the 20% fraction was resuspendedin 1 mL cross-linking buffer. A stock concentration of 50 mM BS3 wasdissolved in 70 μL of UltraPure water per each 8 mg vial. BS3 (60 μl/mLof cells) was added to each resuspended cell fraction, for a finalconcentration of 3 mM BS3, and each cell fraction was mixed gently byswirling. Cell fractions were then incubated on ice for 1 hour, swirlingto mix every 10 minutes. After BS3 incubation, 15 mL quench solution wasadded directly to cells and incubated for 15 minutes at roomtemperature. The cells were centrifuged for 15 min at 1200×g, and thequench buffer was carefully decanted. The pellets were washed 1× withPBS+EDTA as previously described. The cells were then lysed by adding 20mL of lysis buffer (Pierce™ IP Lysis Buffer #87788) to each of the 40%fractions and 10 mL to the 20% fraction and subsequently incubated onice for 15 min. The cell lysates were then centrifuged for 10 min at13,000×g at 4° C.

MabSelect SuRe resin (GE Healthcare Life Sciences, No. 17543801) wasprepared for use in the IP and protein purification of the preparedcells. Sterile Mab Select Sure was equilibrated with 20 column volumes(CV) sterile PBS. One hundred and fifty microliters of Mab Select SuRewas used for the AB102 and positive control samples and 75 μL was usedfor the ISO sample. The sterile and equilibrated MabSelect SuRe resinwas then transferred to 50 mL conical tubes. After centrifugation,supernatant was added to tubes with prepared MabSelect SuRe resin.Samples were incubated overnight with end-over-end mixing at 4° C. Thenext day, samples were allowed to settle on ice for 10 min. MabSelectSuRe resin was transferred by pipet to a disposable drip column. Resinwas washed with 20 column volumes sterile PBS. Prior to elution, thecolumn was placed in a collection tube and centrifuged for 30 seconds(s) at 9,000 rpm in an Eppendorf benchtop microcentrifuge to removeexcess liquid. A stopper was placed on the bottom of the column. Samplewas eluted from the resin by adding one column volume of 50 mM glycinepH 2.5 as elution buffer to the stoppered column and mixing with pipet,then allowing the mixture to incubate at RT for 8 min. (One-tenth of thetotal volume (resin and elution buffer) was removed from each sample andset aside for determination of cross-linking by western blot analysis.See below.) The remaining samples were neutralized by adding 1/10 volumeof 2 M Tris pH 8.0. Eluate was collected by removing stopper from bottomof the column and immediately placing drip column in a 1.8 mL Eppendorfcentrifuge tube. Column assembly was centrifuged for 30 s at 9,000 rpmin a benchtop microcentrifuge unit. Eluate was neutralized by adding1/10^(th) eluate volume of sterile 2 M Tris pH 8.0 to the eluatefraction. Elution protocol was repeated to ensure complete proteinremoval. Eluate fractions were stored at −80° C. until western blotconfirmed cross-linking. Western blot analysis for confirmation of crosslinking

The reserved eluate fractions were mixed with Laemmli Sample Buffer(Bio-Rad, No. 161-0747)+10% 2-mercaptoethanol. Samples were heated at90° C. for 10 minutes. Samples were loaded on a 4-12% Bis-Tris gradientpolyacrylamide gel. The gel was run for 80 min at 200 V. After theloading, the gel was transferred to PVDF western blot membrane and runovernight at 4° C. at 25 V. The next morning the blot was blocked for 3hr at RT with SuperBlock (ThermoFisher Scientific, No. 37515). Afterblocking, the membrane was washed 1× in SuperBlock. The western blot wasprobed with 1:1000 anti-human IgG HRP overnight at 4° C. The followingday, the blot was washed 4× with PBST at 10 min per wash. The westernblot was washed with PBS 1× for 10 min. The western blot was thendeveloped with SuperSignal West Dura Extended Duration Substrate(ThermoFisher Scientific, No. 34076). Upon exposure, the western blotshowed clear super-shifted bands representing positive cross-linking forAB102. Cross-linked bands were also observed for the positive controlantibody. As expected, no molecular weight shift was observed for ISO.

The remainder of the samples were then acetone precipitated, run onSDS-PAGE gels in which the cross-linked bands were then excised andprepared for mass spectrometry evaluation. The eluate samples (AB101,ISO, and positive control from above) were thawed. Four times the eluatevolume of cold (−20° C.) acetone was added to each sample. The sampleswere then vortexed vigorously and incubated at −20° C. for 1 hr. Thesamples were centrifuged for 10 min at 15,000×g. The supernatant wasdecanted, being careful not to disrupt pellet. The pellets were dried inspeed vac for 5 min or until liquid has evaporated. The pellets wereresuspended in 15 μL per tube and mixed until dissolved. Eluates werecombined for each condition. Sample loading buffer+10% 2-mercaptoethanolwas added and the samples were heated for 5 min at 90° C. Fivemicroliters of each sample was reserved for SYPRO Ruby Protein Gel Stain(ThermoFisher Scientific, No. S12000) analysis. The entire remainingvolume of sample was loaded on 4-12% Bis-Tris gradient polyacrylamidegel and run on the gel for 80 min at 200 V. The gel was removed from thecassette and washed 3×10 min with Mass Spectrometry grade water. The gelwas then stained with Safe Stain blue for 1 hr. The stain was decantedand then the gel was destained with mass spectrometry grade water by 2washes at 30 min per wash. Scalpels and tweezers were extensivelysprayed with ethanol (EtOH) before the cross-linked bands were excisedfrom the gel and placed in sterile Eppendorf tubes. The tubes werefilled with enough sterile, ultrapure water to cover gel sections,packed on wet ice, and shipped to MS Bioworks.

The mass spectrometry analysis by MS Bioworks was performed. For themass spectrometry, all of each submitted sample was processed by in-geldigestion with trypsin using a ProGest robot (DigiLab) by first washingwith 25 mM ammonium bicarbonate followed by acetonitrile, reducing with10 mM dithiothreitol at 60° C. followed by alkylation with 50 mMiodoacetamide at RT, digesting with trypsin (Promega) at 37° C. for 4hr, and then quenching with formic acid, digests were pooling andanalyzing without further processing. Each digested sample was thenanalyzed by nano LC-MS/MS with a Waters M-Class NanoAcquity HPLC systeminterfaced to a ThermoFisher Fusion Lumos. Peptides were loaded on atrapping column and eluted over a 75 μm analytical column at 350 nL/min;both columns were packed with Luna C18 resin (Phenomenex, No.00C-4041-E0). The mass spectrometer was operated in data-dependent mode,with the Orbitrap operating at 60,000 FWHM and 15,000 FWHM for MS andMS/MS with a 3 s cycle time. Advanced Peak Determination was enabled. 4hr of instrument time was used/sample. For the data processing by MSBioworks, data were searched using a local copy of Mascot (MatrixScience) with the following parameters: Enzyme: Trypsin/P; Database:SwissProt Human (concatenated forward and reverse plus common potentialcontaminants); Fixed modification: Carbamidomethyl (C); Variablemodifications: Oxidation (M), Acetyl (N-term), Pyro-Glu (N-term Q),Deamidation (N/Q); Mass values: Monoisotopic; Peptide Mass Tolerance: 10ppm; Fragment Mass Tolerance: 0.02 Da; and Max Missed Cleavages: 2.Mascot DAT files were parsed into Scaffold (Proteome Software) forvalidation, filtering and to create a non-redundant list per sample.Data were filtered at 1% protein and peptide FDR and requiring at leasttwo unique peptides per protein. Spectral Abundance Factor (SAF) wasconverted to Normalized Spectral Abundance Factor (NSAF), which was usedto approximate relative abundance of proteins within a given sample, andrelative abundance of a given protein between samples.

Examination of proteins observed in the BS3-cross-linked data from MSBioworks did not support the full list of targets identified in theabove IP involving PFA. Galectin-3 was found in both the heavy and lightdata with greater representation in the light data set). Galectin-1 wasfound in both the heavy and light data with only 3/5 peptides labeledwith the heavy isotope. LILRB2 was not observed. uPAR was not observed.PPIA was found in both the heavy and light data with near equalrepresentation in both. FIG. 6A shows the top 20 targets for AB102. FIG.6B shows the top cell surface targets for AB102. FIG. 6C shows the topcell surface targets for AB102 compared to isotype negative control(ISO). Of the cell surface proteins immunoprecipitated with AB102 usinga BS3 cross-linker, CD163 had the highest spectral abundance factor(SAF). CD163 was immunoprecipitated with AB102 by both the PFA and BS3methods. SAF=spectral counts normalized to protein size (molecularweight) for each target, and SAF values in FIG. 6A and FIG. 6B arevalues for AB102 subtracted from SAF for isotype control for eachtarget.

Example 5—AB101 Antibody in FcNull Framework Immunoprecipitates aGlycoform of CD163

Glycosylation is a highly regulated post-translational modification thataffects the protein conformation, stability and function, and it plays acritical role in establishing protein-protein interactions (i.e., thebinding of ligand to its cognate receptor). The AB102 antibody, and byextension AB101, has specificity for a distinct higher molecular weightglycoform of CD163, which potentially affects CD163 interactions withother proteins necessary for the activity of immune suppressivemacrophages.

The AB101 FcNull antibody comprises AB101 variable domains in an IgG1sequence modified to substantially reduce binding of the antibody to Fcreceptors (Fc null), and is designated as AB102. For analysis by westernblot and SYPRO Ruby Protein Gel Stain, 12 μL of 4×NuPAGE LDS SampleBuffer (ThermoFisher, No. NP0007) with 10% 2-mercaptoethanol was addedto a 25 μL aliquot of PBS-beads from EXAMPLE 4 and incubated at 95° C.for 25 min. Samples were run on 4-12% Bis-Tris gradient polyacrylamidegels. For direct visualization, gels were stained with SYPRO RubyProtein Gel Stain per the manufacturer's instructions. For westernblots, proteins were transferred to a PVDF membrane at 20 V overnight at4° C. The following morning, the membranes were blocked in 5.0 mL ofSuperBlock (ThermoFisher, No. 37515; no Tween 20) for 1 hr. Primaryanti-CD163 antibody (goat IgG polyclonal; R&D, No. AF1607) was added tothe membrane at a concentration of 1 μg/mL. After an approximately 3-hrincubation in primary Ab (rocking at room temp) the membrane was washed3× with approximately 5 mL of PBST, 5 minutes per wash. Followingwashes, a 5 mL of SuperBlock (no Tween 20) containing 1:10,000 (v/v)HRP-conjugated anti-goat F(ab′)₂-specific secondary Ab (various vendors)was added to the membrane and incubated at room temp for approximately 1hr. After incubation with the secondary Ab, the membrane was washed 3×with 5 mL PBST for approximately 5 min per wash. The membrane was imagedusing SuperSignal West Dura Extended Duration Substrate with a FotoDyneAnalyst Luminary Convertible transilluminator FX workstation accordingto the manufacturer's instructions.

As shown in FIG. 7 , AB102 immunoprecipitates a distinct highermolecular weight glycoform of CD163. Both bands in the doublet indicatedby arrows appear to be distinct glycoforms of CD163.

Example 6—AB101 Binds to Human, but not Mouse, Recombinant CD163 Protein

This example shows that AB101 binds to human, but not to mouse,recombinant CD163 protein. AB101 binding to His-tagged recombinant humanCD163 (R&D Systems, No. 1607-CD-050) and recombinant murine CD163 (R&DSystems, No. 7435-CD-050) proteins was determined using ELISA.Recombinant proteins were diluted in PBS to 5 μg/mL and added to384-well High binding ELISA plates (Greiner Bio-One, No. 781061) at 25μL per well and incubated at 4° C. overnight. The plates were washedthree times with PBS, using the BioTek ELx405 Select microplate washer(wash program ELISA_384_PBS_3×_wash) and then blocked with 90 μL/well ofblocking buffer (2% nonfat, dry milk/PBS+0.05% Tween 20) for 1 hr at RT.

After blocking, 25 μL per well of primary antibodies were added to theplates and incubated for 1 hr at room temp. The test antibody was AB101;the control anti-huCD163 antibody was a commercially available antibodyin murine IgG1 framework (R&D Systems, #MAB1607); the isotype controlswere a proprietary mAb, in human IgG1, human FcNull, and murine IgG1frameworks, with known specificity. After primary antibody binding,plates were washed three times with PBS using the EL405× (wash programELISA_384_PBS_3×_wash). The secondary antibody for anti-hu CD163 was thegoat anti-mouse IgG F(ab)′2 HRP (Jackson Immunoresearch, No.115-035-072), and secondary antibody for AB101 and AB102 was goatanti-human IgG F(ab)′2 HRP (Jackson Immunoresearch, No. 109-035-097).Secondary antibodies were diluted to 1:2500 in 2% nonfat, dry milk/PBSand 25 μL per well was added to the respective plate and incubated atroom temp for 1 hr. The plates were washed four times with PBS, usingthe EL405× (Wash program ELISA_384_PBS_4×_wash). After removal of thefinal wash, 25 μL/well of neat Ultra-TMB was added and the plates wereincubated for 10-15 min at room temp, protected from light. Afterdevelopment, the reaction was stopped by adding 25 μL per well of 0.3 MHCl and plates were read using the SpectraMax M5e instrument at 450 nm.

As shown in FIG. 8 , the AB101, AB102, and the control CD163 antibodybound to huCD163 in this assay, while the isotype control showed noappreciable binding. Neither AB101 nor the control anti-huCD163 antibodybound to the recombinant murine CD163, while a commercially availableanti-muCD163 antibody bound as expected (FIG. 9 ).

Example 7—Polyclonal Anti-CD163 Antibody Blocks Binding of AB101 to M2cMacrophages

This example shows polyclonal anti-CD163 antibody blocks binding ofAB101 to M2c cells. To examine the specificity of AB101 binding to CD163on M2c cells, blocking experiments were performed using a commercialpolyclonal antibody to human CD163.

The harvested M2c cells were resuspended (1 million cells per 100 μL) inFACS buffer (PBS containing 2 mM EDTA) (ThermoFisher No. 15575-038) andan appropriate volume of human Fc-block (10 μg of recombinant human Fcblock per million cells) was added. The cells were incubated at RT for20 min. Goat anti-huCD163 polyclonal antibody (R&D, #AF1607) and Goatcontrol polyclonal antibody (Sigma, #PP40) were added at a finalconcentration of 200 μg/mL and incubated at RT for 1 hr. The cells werediluted with FACS buffer to a final volume of 1 million cells/mL and 40μL/well were transferred to v-bottom polypropylene opaque 96-wellplates.

Alexa Fluor® 647 labeled AB101 and APC-labeled anti-huCD163 (R&DSystems, #FAB1607A) antibodies were added, and the cells incubated for90 min at 4° C. The cells were washed by adding 250 μL of FACS buffercontaining 5% BSA to each well using a multidrop, centrifuged at 350×gfor 5 min, and the buffers were removed. The wash step was repeated 2×with wash volume of 300 μL FACS buffer. 50 μL of FACS buffer withviability dye e780 (1:1000 dilution) was added to each well, and theplates incubated for 20 min at RT. The cells were then washed by adding250 μL of FACS buffer to each well using the multidrop, centrifuged at350×g for 5 min, and the buffers were removed. FACS buffer (75 μL) wasadded to each well and sample analysis performed using a BD Canto IIflow cytometer.

The pretreatment of M2c macrophages with polyclonal anti-CD163 antibodyblocked binding of the AB101 antibody (FIG. 10 ) and a controlmonoclonal anti-huCD163 antibody (FIG. 11 ) to M2c macrophages. Thepretreatment of M2c macrophages with goat control polyclonal antibodydid not block binding of the AB101 antibody (FIG. 10 ) and a controlmonoclonal anti-huCD163 antibody (FIG. 11 ) to M2c macrophages.

Example 8—siRNA Knockdown of CD163 in Human M2c Macrophages afterPolarization Reduces Binding of AB102

This example shows siRNA knockdown of CD163 in human M2c macrophagesafter polarization reduces binding of AB102. The methods used in thisexample are based on Troegeler et al., Immunol Cell Biol 92:699-708(2014).

Isolated monocytes (from three donors) were separately plated at 1.5×10⁶cells per well in 6-well plates. On Day 4, the medium was removed andfresh X-VIVO medium containing 10% FBS, 100 ng/ml M-CSF (Peprotech No.300-25), and 50 ng/mL IL-10 (Peprotech No. 200-10) was added to eachwell. On Day 6, the medium was removed, and the cells were washed twicewith warm X-VIVO medium containing 10% FBS. One milliliter of X-VIVOmedium+10% FBS was added to each well and cells were returned to theincubator while the siRNA transfection solutions were prepared.

The following human ON-TARGETplus siRNA—SMARTpool Reagents (Dharmacon)were tested: CD163 (Cat. No. L-007847-00-0005); SCRAM (Cat. No.D-001810-10-05); CD206 (Cat. No. L-011730-00-0005); CD163L1 (Cat. No.L-008024-00-0005); PPIA (Cat. No. L-004979-04-0005); FCGR2A (Cat. No.L-014152-00-0005); FCGR3A (Cat. No. L-016308-00-0005); LGALS1 (Cat. No.L-011718-00-0005); LGALS3 (Cat. No. L-010606-00-0005); LILRB2 (Cat. No.L-020017-00-0005); FCGR2C (Cat. No. L-027340-02-0005); and UPAR (Cat.No. L-006388-00-0005).

To prepare the siRNA transfections, ON-TARGETplus SMART siRNA pools wereused to make 200 μM master stocks. Lyophilized oligonucleotides (5 nmoleach) were resuspended in 25 μL of 1× Thermo siRNA buffer (Thermo No.B002000). Aliquots were stored at −80° C. These master stocks were thendiluted with cell grade ultrapure water (Hyclone No. SH3052902) to make20 μM working stocks. To make the master mix (enough for 6 wells of a6-well plate), the following reagents were mixed: 120 μL 20 μM siRNApool (final concentration 200 nM), 270 μL of HiPerFect (Qiagen No.301705), and 2.64 mL warm RPMI (with no FBS or other additives). Themaster mix for siSCRAM (scrambled siRNA) contained the followingamounts: 720 μL 20 μM siSCRAM, 1.23 mL HiPerFect, and 12.1 mL RPMI. Themixtures were combined and incubated at RT for 15 min, with periodicmixing by inversion. Just before use, tubes were centrifuged briefly,and the siRNA mix (495 μL/well) was added dropwise to cells. Plates wererocked gently to mix and then incubated at 37° C. for 6 hr. Followingincubation, 2 mL of X-VIVO medium containing 10% FBS and IL-10 (finalconcentration 50 ng/mL) and M-CSF (Peprotech No. 300-25; finalconcentration 50 μg/mL) was added. The following day the medium waschanged (keeping in IL-10 and M-CSF) to remove transfection reagent andany dying cells.

On day 8, the macrophages were either lifted and stained with antibodiesfor flow cytometry or lysed for RT-qPCR.

For those macrophages used for flow cytometry, the following method wasused. siRNA-treated M2 macrophages were harvested with macrophagedetachment solution, incubated in detachment solution at 4° C. for 45min, and then gently scraped off the plates. The macrophage detachmentsolution was replaced with cold PBS−/− (PBS without calcium andmagnesium; Hyclone No. SH30028.02) containing 0.2 mM EDTA and 0.1% HSA.The cells were spun down at 650×g for 5 min and then resuspended in 0.5mL cold Block solution (FACS buffer+10% NGS+10 μg of human IgG Fcfragment protein (Abcam No. ab90285)) per million cells. The cells werecounted and an average of 1×10⁶ per mL was used for furthercalculations. The cells were incubated at RT for 15 min (to increase FcRbinding), followed by a 30 min incubation on ice for full blocking. Analiquot of unstained cells was set aside for compensation controls. e780viability dye (ThermoFisher eBioscience No. 65-0865-18) was added to theremainder at a final dilution of 1:1000. Cells were aliquoted into96-well plates (40 μL per well), and 10 μL of antibody solution wasadded to each well. Antibodies were prepared with a startingconcentration of 100 μg/mL in FACS buffer and serially diluted in FACSbuffer. After addition of antibodies, each set of cells was tested withan antibody panel (AB101 in FcNull framework conjugated to AF647 andcommercial anti-CD163 conjugated to BV421 [BioLegend No. 333612]) and anisotype panel (ISO2 in FcNull framework conjugated to AF647 andcommercial mIgG1 conjugated to BV421). Final antibody concentrationranged from 20 μg/mL to 0.3 μg/mL. Cells were incubated in primaryantibody for 1 hr at 4° C., then pelleted at 450×g and washed threetimes with 150 μL PBS-EDTA. After pelleting, cells were then resuspendedin 100 μL 4% paraformaldehyde (in PBS−/−) and incubated at RT for 15 min(protected from light). After fixation, cells were spun down at 650×gfor 5 min and washed once with PBS-EDTA. Cells were resuspended in 100μL PBS-EDTA and stored at 4° C. over the weekend (protected from light).Cells were then analyzed by flow cytometry on a BD Canto II machine.

A second set of cells was harvested for use in RT-qPCR assays, asfollows. Cells were harvested in buffer RLT and RNA was isolated usingthe Qiagen RNeasy kit (Qiagen No. 74104), including QIAshredders (QiagenNo. 79654). After elution, RNA was used to make cDNA using theSuperScript III first-strand synthesis system (Invitrogen No. 18080051).To 1.5 μg RNA, RNase-free water was added to a final volume of 10 μL.Added to this was 5 μL of DnaseI master mix ((per sample)=1.5 μL 10×DnaseI buffer+2 μL RNase-free water+1.5 μL DnaseI enzyme). Samples weremixed well and incubated at room temp for 15 min. After incubation, 1.5μL EDTA solution was added to stop the reaction, the samples incubatedat 65° C. for 10 min (to kill the enzyme), and then returned to ice tocool down. dNTPs (1.5 μL) and oligo-dT (1.5 μL) were added to eachsample and mixed well. The samples were incubated at 65° C. for 5 minand then returned to ice for 2 min. After samples were chilled, 13 μL ofsample was removed to fresh tubes and +RT master mix (7 μL) was added toeach. To the remaining 7.5 μL of sample, −RT master mix (3.5 μL) wasadded for the negative control samples. For the +RT master mix (persample): 4 μL 5× First strand buffer, 1 μL 0.1 M DTT, 1 μL RNaseOUT, and1 μL SuperScript III reverse transcriptase enzyme (all part of theSuperScript III system referenced above). For the −RT master mix, theSuperScript III enzyme was replaced with RNase-free water.

Samples were mixed well and incubated at 25° C. for 5 min. Afterincubation, the samples were incubated at 50° C. for 30 min, followed byincubation at 55° C. for 30 min, and then incubation at 70° C. for 15min. Samples were then cooled to 4° C. before proceeding and kept onice. Before use, 20 μL of RNase-free water was added to the +RT samplesand 10 μL of RNase-free water added to the −RT samples to dilute thecDNA for RT-qPCR.

For the qPCR, 2 μL of diluted cDNA was mixed with 0.2 μL of each primer(10 μM stock concentrations), 2.6 μL RNase-free water, and 5 μL of2×SYBR Green qPCR mix (BioRad No. 1725271). Standard curves were madeusing a dilution series of the untreated control cells. Samples were runon the StepOne qPCR instrument using the default settings, including atemperature melt curve. Samples were normalized to amplification ofRpl17a as an internal control.

As shown in FIG. 12 , (representative of the three replicates) treatmentof polarized M2c macrophages with siRNA to CD163 substantially reducedbinding of the AB102 antibody compared to the scrambled siRNA (siSCRAM)or no siRNA treated M2c macrophages.

Example 9—AB102 Binding to Polarized Human M2c Macrophages after siRNAKnockdown

This example shows AB102 binding to polarized human M2c macrophagesafter siRNA knockdown using various siRNAs.

Monocytes were isolated from whole blood, plated at 1.5×10⁶ cells perwell (6-well plates), and cultured under M2-polarizing conditions asdescribed in EXAMPLE 8, above. On day 6, medium was changed and 100ng/mL M-CSF and 50 ng/mL IL-10 were added.

siRNA treatment was done on day 8 with various siRNA as describedpreviously (see EXAMPLE 8). The following day the medium was aspiratedto remove the transfection reagent and any dying cells, and fresh mediumwas added (still containing IL-10 and M-CSF). On day 10, FACS analysiswas performed on siRNA-treated cells, and a second set of cells washarvested in buffer RLT for RT-qPCR (see EXAMPLE 8). The data wasnormalized, using AB102 binding geometric MFI to siSCRAM treated M2c as100%, and isotype control antibody binding geometric MFI to untreatedM2c as 0%.

siRNA knockdown of CD163 reduced binding of the AB102 antibody. Incontrast, no evidence was seen of reduction in AB102 binding afterknockdown with siCD206; siCD163L1; siPPIA; siLGALS1; siLGALS3; siLILRB2;or siUPAR, except some slight decrease with siRNAs against FCGR2A+FCGR3A(in 1 out 3 donors), FCGR2C, or FCGR3A as shown in FIG. 13 . In fact,the binding intensity of AB102 increased in several of the siRNAconditions. The RT-qPCR showed strong knockdown of the targets.

Example 10—LPS-Induced Decrease of CD163 Expression on the Cell Surfaceof M2c Macrophages Reduces AB101 Binding to Macrophages

This example shows that reduced binding of AB101 to M2c macrophagesafter LPS-induced shedding of CD163. Monocytes were isolated asdescribed in EXAMPLE 2 and plated at 1×10⁴ cells/well in flat bottom,tissue culture treated 96 well plates in X-VIVO medium containing 10%FBS, 50 ng/mL M-CSF and 50 ng/mL IL-10 at 100 μL/well. On day 7, half ofthe cells were treated for 24 hr with 10 ng/mL LPS (lipopolysaccharidesfrom E. coli O111:B4; Sigma No. L5293-2ML). Cells were labeled withtitrations of unconjugated primary antibodies, anti-CD163 (R&D SystemsMAB1607-100) and AB101, according to methods previously described, usingeight serial 5-fold dilutions of each, starting at 200 nM. Alexa Fluor®647 AffiniPure F(ab′)2 Fragment Goat Anti-Human IgG, F(ab′)2 fragmentantibody was used as the secondary (Jackson ImmunoResearch 109-606-006).Cells were analyzed by flow cytometry.

As shown in FIG. 14 , treatment of the cultured M2c macrophages with LPSresulted in a loss of binding by both AB101 antibody and the controlanti-CD163 antibody.

Example 11—AB101 Blockage of Myeloid Cell Suppression of T CellActivation (IL-2 Production) and Proliferation

This example shows AB101 blocks myeloid cell suppression of T cellactivation (IL-2 production) and proliferation.

To assess the ability of to relieve the M2 macrophage-mediatedsuppression of T cell activation, M0, M1 and M2c macrophages weregenerated from human monocytes. M0 macrophages were cultured with AB101or Isotype control under three treatment protocols: 1) In the presenceof AB101 (or isotype control antibody) during polarization from M0 toM2c macrophage (Day 5-7, “pre”-condition), 2) in the presence of AB101(or isotype antibody) post polarization (Day 7 onward,“post”-condition), or 3) conditions 1 and 2 combined (“pre” and “post”polarization).

Generation of M0 macrophages. At Day 0, monocytes from individual donors(isolated as described in EXAMPLE 2) were plated at 2.5×10⁵ cells/wellof a 96-well tissue culture plate in M0 medium (X-VIVO medium+10%FBS+100 ng/mL M-CSF), and incubated at 37° C., 5% CO₂ for 5 days.

Polarization of M0 macrophages to M1 or M2c macrophages. 5-day old M0macrophages were polarized to M2c by culturing the cells in M0 medium100 ng/mL IL-10 (Peprotech No. 200-10), and to M1 by culturing in M0medium+100 ng/mL IFN-gamma (Peprotech No. 300-03). For cells treatedwith AB101 or IgG1 isotype control, those antibodies were added at 20ng/mL in M2c medium. At Day 6, for M1 macrophages, medium was discardedand fresh M0 medium+100 ng/mL IFN-gamma+1 ng/mL LPS (lipopolysaccharidesfrom E. coli O111:B4; Sigma No. L5293-2ML) was added.

PBMCs from autologous donors were used to isolate CD8⁺ T cells (asdescribed in EXAMPLE 2). T cells were plated into T75 flasks overnightin X-VIVO+10% FBS until the day of co-culture with macrophages (Day 7).

CellTrace™ Violet Proliferation Dye kit (ThermoFisher No. C34557), whichallows tracing of multiple generations using dye dilution by flowcytometry, was used to stain T cells prior to co-culture. CellTrace™staining was performed according to manufacturer's protocol.

At Day 7, supernatant was removed from plated macrophages, and mediumwas replaced with 100 μL of X-VIVO medium+10% FBS+0.5 μg/mL OKT3.Macrophages were incubated at 37° C., 5% CO₂ for 1 hr. T cells wereharvested from flasks and resuspended at 115,000 T cells in 100 μL/well(1.15 million/mL) in flat bottom 96 well plates in the absence orpresence of AB101 (20 μg/mL) or isotype control (20 μg/mL) for“Pre/Post-polarization” and “post-polarization” treatments. T cells wereadded to macrophages at a volume of 100 μL, to give a final volume of200 μL/well and final concentration of 0.25 μg/mL OKT3. Plates wereincubated at 37° C., 5% CO₂ for 24 hr. On Day 8, supernatants werecollected. IL-2 levels were measured using a CisBio HTRF IL-2 kit (No.62HIL02PEG) according to the manufacturer's protocol, with the followingmodifications: the assay was performed in low-volume 384-well plates(Greiner Bio-One No. 784075); all volumes were halved; and the plateswere briefly spun to bring bubbles to the surface.

As shown in FIG. 15 , AB101 antibody blocked the ability of myeloidcells to suppress T-cell activation, as evidenced by increased IL-2production, a marker of T-cell stimulation and proliferation. Cells weretreated with AB101 or isotype control during polarization (with IL-10),the “Pre” condition.

On Day 10, co-cultured T cells from each 96-well plate were transferredto a V-bottomed 96-well plate (ThermoFisher No. 249946), and pelleted bycentrifuging at 300×g for 2 min. Pellets were resuspended in 100 μL ofe780 viability dye (eBiosciences No. 65-0865-14) in PBS (0.5 μL/mL) andincubated for 10 min at RT in the dark.

Following e780 staining, cells were washed by adding 150 μL FACS Buffer(1×PBS+2 mM EDTA+1% FBS) and centrifuged at 300×g for 2 min. Supernatantwas removed. Cell pellets were resuspended in 50 μL/well FACS Block(human TruStain FcX™ [Fc receptor blocking solution; BioLegend No.422302] at 5 μL/100 μL in FACS Buffer), and incubated for 30 min at 4°C.

Antibody cocktails (2×) were made using FACS Block containingAPC-labeled anti-CD8 (BD Pharmingen No. 561953) at a 1:50 dilution(Final concentration is 1:100); and FITC-labeled anti-CD14 at a 1:50dilution (Final conc is 1:100) (BD No. 347493). This antibody cocktailwas added at 50 μL/well and incubated for 30 min on ice in the dark. Thestain was washed with 150 μL/well FACS Buffer. Cells were pelleted at300×g for 2 min. Supernatant was removed, and cells were fixed in 25 μLof 4% PFA for 15 min on ice in the dark. Before analysis on a flowcytometer, 75 μL/well of PBS was added.

As shown in FIG. 16 and FIG. 17 , the AB101 antibody permittedOKT3-induced CD4⁺ and CD8⁺ T cell proliferation with AB101 treatmentduring polarization, respectively. Furthermore, as shown in FIG. 18 ,treatment with AB101 antibody post-polarization, during co-culture withCD3⁺ T cells (labeled “post” on graph), or combined during andpost-polarization (labeled “Pre and Post” on graph) resulted in enhancedIL-2 production, when compared to isotype antibody treatment. Theseresults indicate that the binding of AB101 to M2c macrophages relievesM2c-mediated suppression of T cell proliferation and IL-2 production.The AB101 treatment is effective during polarization with IL-10,overcoming a constitutively suppressive signal, and after M2cpolarization, which is representative of the suppressive TAMs in thetumor microenvironment in vivo.

Example 12—Reduction of M2c Surface Marker Expression

This example shows the reduction of M2c surface maker expression aftertreatment with AB101.

Monocytes were isolated (as described in EXAMPLE 2) and polarized to M2cmacrophages (as described in EXAMPLE 11) in the presence of AB101 or theisotype control antibody. Cells were then stained for surface markerexpression of phenotyping antibodies following the protocols describedin EXAMPLE 11. Normalized Median Fluorescence Intensity (MFI) isdisplayed in the graph below for macrophage surface expression followingthe treatment of M2c macrophages with AB101 or with the isotype controlantibody. Live cells were gated using e780 fixable viability dye.Normalized MFI was calculated by dividing the MFI of the AB101 treatedcells by the MFI of M2c cells treated with isotype control. Samples werethen normalized to percent of M2c control, to show relative change insurface marker expression. Data from 7 donors was averaged andstatistics were performed using 2-way ANOVA.

As shown in FIG. 19 , “Pre” treatment of M2 macrophages with the AB101antibody during polarization reduced expression of CD16, CD64,Calreticulin, and Siglec-15. CD16 (FcγRIIIa), a low-affinity IgGreceptor, is highly expressed on M2 suppressive macrophages. CD64(FcγRI), a high-affinity IgG receptor, is also highly expressed on M2s.Siglec-15 is an ITIM-containing transmembrane protein involved in immunesuppression, which is specifically expressed on suppressive macrophages.

No change in these markers was seen in M2c cells treated with isotypecontrol antibody. Similarly, surface expression of PD-L1, CD11b, CD14,CD32, CD163, CD206, HLA-DR, CD204, CD33, CD80, CD86, HLA-DR, DP, DQ,CD48, MARCO, LILRB2, CD172a (SIRPα), IL10R, and IL18R were evaluated,but no change in expression of these markers was observed with AB101treatment.

Example 13—AB101-Treated M2c Macrophages Skew OKT-3 Activated T CellsTowards a Th1 Phenotype

This example shows that AB101-treated M2c macrophages induce theexpression of Th1-associated surface markers by OKT3-stimulated T cells.The data suggests that AB101 treatment of M2c cells inhibitsM2c-mediated immune suppression and modulates the activation ofanti-tumor Th1 cells.

Myeloid cells in the tumor microenvironment, tumor associatedmacrophages (TAMs), have been shown to orchestrate a dampened immuneresponse which facilitates tumor grown. Often, this effect can be seenas skewing T cells to a lower ratio of Th1/Th2 (e.g., skewing T cells toa Th2 phenotype). Therefore, we hypothesized that AB101 will affect thecross talk between the TAMs and tumor infiltrating lymphocytes (TILs),relieving the suppressive effect of the TAMs on the TILs.

The ratio of Th1 to Th2-helper cells was assessed in the presence orabsence of AB101 and isotype control. M2c macrophages were treated withAB101 or isotype control on Day 5 (“Pre”=during polarization), and Day 7(“Post” polarization, during co-culture). Starting on Day 7, treated M2cmacrophages are co-cultured with OKT3 stimulated CD3⁺ T cells for 3 daysto allow for T cell proliferation. Following T cell proliferation, onDay 10, T cells were removed from co-culture and stained with cellsurface marker antibody panels to determine ratio of Th1 to Th2 skewing.Following surface marker and cell viability staining, T cells were fixedand analyzed for presence of Th1 or Th2 markers by flow cytometry. Panel1 was used to determine ratio of Th1/Th2, Th17, and Treg, while panel 2was used to determine T cell activation and exhaustion.

Monocytes were obtained and cultured to macrophages, and the macrophageswere polarized as described in previous examples.

CD3⁺ T cells were obtained as described in EXAMPLE 2, using the StemCellCD3⁺ negative selection kits according to the manufacturer'sinstructions.

The macrophages and T cells were co-cultured, as described in EXAMPLE11, for three days.

At Day 10, cells were labeled using antibodies cocktails as set forth inthe Table 4 below according to the method described in EXAMPLE 11.

Antibody cocktail is made at 2× using remaining 50 μL/well of Blockingbuffer, with Panel 1 antibodies at 1:50 (Final conc is 1:100), and Panel2 antibodies at 1:50 (Final conc is 1:100)

TABLE 4 Antibody Cocktail Mixes for assessing Th1/Th2, Th17 (Panel 1)and Exhaustion/Activation (Panel 2) of T cells. Antibody Panel 1:Antibody Panel 2: Th1/Th2, Th17 Exhaustion/Activation Surface CD4 - PE(BD Pharmingen No. 55347) CD4 - PE (BD Pharmingen No. 55347) markersCD69 - PE-Cy7 (BioLegend No. CD8 - APC (BIOLEGEND No. 344721) 104511)LAG3 - BV421 (BioLegend No. 369313) CD25 - APC (BioLegend No. 101909)OX40 - BV510 (BioLegend No. 745040 CD127 - BV510 (BD BIOSCIENCE PD-1 -PerCP-Cy5.5 (BioLegend No. NO. 563086) 135207) CXCR3 - PerCP-Cy5.5(BioLegend No. ICOS - PE-Cy7 (BioLegend No. 329805) 126513) CTLA4 - FITC(eBioscience 11-1529-42) CD194 (CCR4) - BV421 (BioLegend No. 359413)CD196 (CCR6) - BV510 (BioLegend No. 353423) Marker Th1: CD4⁺, CD69⁺,CD196⁻, CXCR3⁺, Activated: ICOS⁺, OX-40⁺ Identification CCR4⁻ Exhausted:LAG-3⁺, PD-1⁺, CTLA-4⁺ Th2: CD4⁺, CD196⁻, CXCR3⁻, CCR4⁺ Th T cells: CD4⁺Treg: CD4⁺, CD25⁺, CD127⁻ Tc T cells: CD8⁺ Th17: CD4⁻, CD196⁺, CXCR3⁻,CCR4⁺

The in vitro myeloid cells, M2c cells, had immunosuppressive effects onactivated T cells in co-culture, in which the M2c inhibited T cellproliferation and skewed T cells to a Th2 phenotype.

Treatment with AB101 alleviated the suppressive effects of the M2ccells, resulting in the ability of the stimulated T cells to produceIL-2, proliferate (as shown in EXAMPLE 11), and be skewed them toward anactivated Th1, pro-inflammatory, phenotype. FIG. 20 shows that treatmentof M2c cells with AB101 increased the Th1/Th2 ratio compared to theisotype control, indicating that AB101 treatment of the M2c cells causedT cells to skew toward the Th1 phenotype. Furthermore, FIG. 21 showsthat treatment of M2c cells with AB101 increased the expression of CD69on CD4⁺ T cells compared to the isotype control, indicating that AB101treatment of the M2C cells caused the CD4⁺ T cells to skew toward theTh1 phenotype when M2c cells were treated with AB101. FIG. 22 and FIG.23 show that treatment of M2c cells with AB101 increased the expressionof ICOS and OX40, respectively, on CD4⁺ T cells compared to the isotypecontrol, indicating that AB101 treatment of the M2c cells caused theproliferated CD4⁺ T cells to have enhanced expression of activationmarkers.

Example 14—Reduction of Myeloid Cell Suppression of CD19-CD3 BispecificT Cell Engager-Mediated Killing of Raji Cells by CD8 T Cells

This example shows AB101 treatment reduces myeloid cell suppression ofCD19-CD3 BiTE-mediated killing of Raji cells by CD8 T cells. Tumor cellkilling was evaluated for AB101 against an isotype control antibodyusing a Bispecific T cell Engager (BiTE) antibody (bispecific antibodyagainst human CD19 and human CD3; InvivoGen Bimab-hcd19cd3). M2cmacrophages were treated with AB101 or isotype control on Day 5 during(“Pre”) and on Day 7 after (“Post”) polarization, during co-culture.Co-culture with T cells continued for 3 days starting on Day 7 to allowfor T cell proliferation. On Day 10, T cells were removed fromco-culture with macrophages and subsequently incubated onto tumorcells+/−BiTE antibody to facilitate contact between the CytotoxicT-lymphocytes (CTL) and tumor cells. Following treatment with BiTEantibody, tumor cells were stained for viability by flow cytometry.

Monocytes were cultured, and macrophages polarized as described inEXAMPLE 11. CD8⁺ T cells were obtained as described in EXAMPLE 11.Macrophages (25,000 cells/well) were co-cultured with CD8⁺ T cells(115,000 cells/well), using the described method, for three days.

On Day 10, Raji (ATCC No. CCL-86) and K562 (ATCC No. CCL-243) cells werestained with CellTrace Violet using the method described in EXAMPLE 11.The tumor cells were then resuspended in M0 medium at 100 k cells/wellin a flat-bottom 96-well tissue culture plate. Some unstained andstained cells were set aside for single stain control for flow analysis.

On Day 10, CD8⁺ T cells were isolated from T cell/macrophage co-cultureusing a StemCell CD8 Negative selection kit. The recovered T cells wereplated into the Raji and K562 cell plates, 100 μL per well. Bispecificantibody was added to each Raji and K562 plates at final concentrationof 10 ng/mL in final volume of 220 μL/well (some wells without BiTE ascontrols). Cells were cultured in BiTE treatment overnight at 37° C., 5%CO₂.

On Day 11, cells were placed into new V-bottom plate, and centrifuged at300×g for 2 min. Supernatant was collected from all plates andtransferred to new V-bottom 96-well plates, which were then sealed andstored at −80° C. for later cytokine analysis. Cells were resuspended inFACS buffer (PBS+1% FBS), and stained with anti-CD8, anti-CD14 (toexclude non-target cells), and e780 viability dye, as described inEXAMPLE 11. Following staining, cells were rinsed with FACS buffer,fixed using 4% PFA, and resuspended in PBS for flow cytometry analysis.CellTrace violet-labeled tumor cells were evaluated for tumor cell deathby inclusion of Fixable Viability Dye eFluor™ 780 within cells. Cellspositive for eFluor™ 780 dye were plotted as percent dead compared to NoBiTE control wells.

Treatment with AB101 relieved the suppressive effects of the M2cmacrophages, which allowed for increased T cell proliferation comparedto isotype control. Increased CTLs in the presence of BiTE resulted inincreased Raji tumor cell killing, compared to isotype control, as shownin FIG. 24 . K562 was used as a negative control and showed no increasein killing+BiTE antibody.

Example 15—Antibody Internalization by Human Primary M2c Macrophages

At Day 0, monocytes (see EXAMPLE 2) were plated in optically clearbottom, 96-well tissue culture plates at 1×10⁵/well in 100 μL (1×10⁶/mL)of M0 medium to differentiate to macrophages. At Day 5, the plates wereswirled to dislodge floating cells and the medium was gently aspirated.Macrophages were polarized to M2c in 100 μL/well M2c medium as describedin previous examples.

At Day 6, antibodies were labeled using Alexa Fluor™ 647 AntibodyLabeling Kit (Invitrogen No. A20186). Each antibody (100 μg) was dilutedto 2 mg/mL in 50 μL PBS. The antibodies tested were as follows: AB101huIgG1 and AB102 huIgG1 ADCC-Null; CD163 mouse monoclonal IgG1 antibody(R&D Systems MAB1607-100); and Isotype control: ISO1 huIgG1 or ISO2human Fc-null framework.

The entire vial of A-647 carboxylic acid succinimidyl ester from the kitwas resuspended in 150 μL PBS. Aliquots (50 μL) of A-647 solution wereadded to each tube of diluted antibody, now at 1 mg/mL, and the mixturesincubated at RT for 45 min in the dark.

Zebra desalting columns (Thermo 87766) were washed, by first snippingthe bottom and centrifuging for 1 min at 4,100 rpm to remove storagebuffer. Then the columns were washed twice with 300 μL PBS (spin 1 min.at 4,100 rpm), and once w/ 300 μL PBS (spin 4,100 rpm for 2 min.). Thecolumns were placed into new amber tubes and the antibodies wereindividually loaded. The columns were then centrifuged for 2 min at4,100 rpm to elute the Alexa-647 labeled antibodies at 1 mg/mL.

At Day 7, FBS-containing medium was removed from the culture plates byflicking and the plates washed twice with 250 μL cold PBS, followed byaddition of 90 μL of X-VIVO medium either containing 20 μg/mL unlabeledISO1 IgG1 antibody to block Fc receptors, for staining with labeledAB101, ISO1, and anti-CD163 antibody (R&D Systems MAB1607-100), ormedium without unlabeled ISO1 block for staining with AB102 and ISO2.Cells were incubated for 30 min at 37° C., 5% CO₂, followed by additionof labeled antibodies were at a final concentration of 5 μg/mL. After 1hr of incubation, medium was removed and the cells washed with 250 μLcold FACS Buffer, followed by addition of 4% PFA (BD Cytofix/CytopermNo. 554722) for 10 min at RT in the dark. Counterstains for cellularcomponents were prepared, by adding 2 drops/mL of NucBlue™ (MolecularProbes No. R37605) and ActinGreen™ (Molecular Probes No. R37110) per mLto 1× Perm Buffer (BD Perm/Wash No. 554723, diluted 1:10 in water). Theplates were flicked to remove fix and counterstain solutions (20μL/well) were added. Staining proceeded at RT in the dark for 20 min.The cells were then washed by adding 250 μL/well PBS, which was removedand replaced with 50 μL/well PBS. Cells were imaged using a Cellomicsinstrument.

These data are mean fluorescence values inside the cell as determined bycellomics (mean ring average intensity in AF647). The cell is definedand detected by a DAPI stained nucleus (NucBlue) and a FITC labeledcytoskeleton (AcinGreen). FIG. 25 shows representative results of atleast 4 individual donors. In all cases, the isotype control antibodiesdid not internalize and the AB102 antibody internalized to the sameextent as the commercial anti-CD163 antibody (R&D Systems MAB1607-100).AB101 (IgG1) antibody was internalized approximately 2-fold more thaneither AB102 (FcNull) or the commercial CD163 antibody.

Example 16—AB101 Inhibits Tumor Growth in a Human Lung Cancer XenograftModel

AB101 was tested for tumor growth inhibition in vivo in a human lungcancer xenograft model. Following AB101 treatment, tumor size and weightwere significantly reduced compared to control group, with acorresponding increase in the proportion of CD8⁺ T cells as well assurface expressions of T cell activation markers, ICOS and OX40, on CD8⁺T cells in the spleen. No differences were observed for CD4⁺ T cells.These results suggest that AB101 promotes CD8⁺ T cell activation andproliferation, consistent with in vitro studies shown in previousexamples. Furthermore, the proportion of CD11b⁺ cells was increased.CD11b is present on monocytes, macrophages, granulocytes, dendriticcells, and natural killer cells. Taken together, these findings suggestthat AB101 may have therapeutic application to augment the immuneresponse to control tumor burden.

To determine the therapeutic potential of AB101, the effectiveness ofthe AB101 in reducing tumor growth in vivo was tested using the NSG-SGM3mouse strain (The Jackson Laboratory), which supports engraftment ofhuman CD34⁺ hematopoietic stem cells and the reconstitution ofmultilineage immune cell populations.

Frozen aliquots of A549 (human lung carcinoma, p53 wild type) andNCI-H1975 (human lung adenocarcinoma, p53 mutated, p.R273H;) werepurchased from ATCC (cat #CCL-185 and CRL-5908, respectively). A549cells were grown in F-12K medium (ATCC No. 30-2004) supplemented with10% fetal bovine serum (FBS; Corning, cat #35-010-CV) and 1%penicillin-streptomycin (pen/strep, HyClone, cat #SV30010). H1975 cellswere cultured in RPMI (HyClone, cat #SH30096.02) with 10% FBS and 1%Pen/Strep. Cells were expanded at 37° C./5% CO₂ for multiple passagesprior to subcutaneous injection into mice.

NSG-SGM3 mice were transplanted with two human cord blood units,performed by The Jackson Laboratory as previous described Shultz et al.,Nat Rev Immunol 7(2):118-30 (2007) [PubMed: 17259968]; Shultz et al.,Nat Rev Immunol 12(11):786-98 (2012) [PubMed: 23059428]; Ishikawa etal., Curr Top Microbiol Immunol 324:87-94 (2008) [PubMed: 18481454];Pearson et al., Curr Protoc Immunol; Chapter 15: Unit 15.21 (2008)[PubMed: 18491294]. Two of these mice became sick and were euthanized.Upon arrival to the facility, these mice were allowed to acclimate for 5days. The right and left flanks of each mouse were shaved on day 6.

On day 7, A549 and H1975 cells were harvested from the cultures, washed3 times with PBS (phosphate-buffered saline without Ca²⁺ or Mg²⁺;HyClone No. SH30028.02) and resuspended in Corning Matrigel® membranematrix (Fisher Scientific No. CB-40234C) at a density of 5×10⁶ cells/mL.A549 cells were injected into the right flank while H1975 cells wereinjected into the left flank of each mouse at a dose of 5×10⁵ cells in100 μL Matrigel.

Five days post injections, the tumors were measured by digital caliper(Fisher Scientific No. NC0649232). Once tumors reached 50-75 mm3 (tumorvolume=(W (2)×L)/2), the mice were then randomized using a web-basedrandomizer application (https://www.randomizer.org/) and divided into 2groups (shown below) with 7 mice per group:

(1) Isotype control antibody (ISO1 Hu IgG1);

(2) AB101 antibody (Hu IgG1);

The mice received antibody treatments starting on the day ofrandomization and every three days thereafter. Each mouse received 200μg of isotype control or of AB101 per treatment in 100 μL of PBS viaintraperitoneal injection. Tumor size was measured on Mondays,Wednesdays, and Fridays until day 26. Any mice showing signs of fatalmorbidity were documented and euthanized immediately. Mice weresacrificed on day 26. Tumors and spleen were harvested for furtheranalysis.

The isolated tumors were weighed and processed by removing fat, fibrous,and necrotic areas and cutting into 2-4 mm pieces. The processed tumorswere added to a gentleMACS C tube (MACS Miltenyi Biotec, No.130-096-334) containing tumor dissociation enzyme mix solution (TumorDissociation Kit, Mouse; MACS Miltenyi Biotec, No. 130-096-730). Thecells were dissociated using a gentleMACS dissociator (MACS MiltenyiBiotec, No. 130-093-235) and then incubated at 5% CO₂ and 95% humidityfor 30 min. The cells were pelleted, resuspended in PBS and strainedusing a 100-μm cell strainer (Corning No. 352360). Dissociated singlecells were analyzed by flow cytometry.

Spleens were processed and dissociated into single cells by pressingthrough a cell strainer. A 10-mL syringe plunger head was used to removeany fat and fibrous tissues. Splenic cells were pelleted and resuspendedin PBS for analysis by flow cytometry.

Myeloid and T cells from the tumors and spleens were quantified usingflow cytometry with antibody cocktail panels shown in Table 5 below.Cell viability was assessed using e780 viability dye (eBiosciences No.65-0865-14; 1:500 in PBS). The cells were incubated for 10 min at 4° C.with e780 in FACS Buffer (PBS+1% FBS+1 mM EDTA (Fisher Scientific No.15575-038)) prior to staining with primary or isotype controlantibodies. The cells were then washed with 200 μL of FACS buffer, andblocked in 25 μL Fc Block (FACS buffer+5 μL/mL of Fc Block (BioLegendNo. 422302)) at 4° C. for 30 min. The antibody stains (see Table 5below) were added (25 μL) to the cells and incubated at 4° C. foranother 30 min in the dark. Cells were washed 3× before FACS analysis.

TABLE 5 Antibody Cocktail Panels Source Source Number Myeloid Panel 1Anti-CD11b-APC BD Biosciences 550019 Anti-CD16 - PE BioLegend 302007Anti-CD64 - Pacific Blue BioLegend 305018 Anti-HLA-DR - PerCP-Cy5.5BioLegend 361710 Anti-CD80 - FITC BioLegend 305206 Anti-LILRB2 - PE-Cy7BioLegend 338711 Myeloid Panel 2 Anti-CD11b-APC BD Biosciences 550019Anti-CD86 - BV510 BD Biosciences 563697 Anti-PD-L1 - BV421 BioLegend329714 Anti-CD83 - PE-Cy7 BioLegend 305325 Anti-CD163 - FITC BDBiosciences 3563697  Anti-ICOSL - PE BioLegend 309403 T cell Panel 1Anti-CD4-FITC BioLegend 300505 Anti-CD194 (CCR4) - BV421 BioLegend359413 Anti-CD196 (CCR6) - BV510 BioLegend 353423 Anti-CXCR3 -PerCP-Cy5.5 BioLegend 126513 Anti-CD69 - PE-Cy7 BioLegend 104511Anti-CD25 - APC BioLegend 101909 Anti-IL-7Ra - PE BioLegend 135013 Tcell Panel 2 CD8-APC BD Biosciences 561953 Anti-LAG-3 - BV421 BioLegend369313 Anti-OX40 - BV510 BD Biosciences 745040 Anti-PD-1 - PerCP-Cy5.5BioLegend 135207 Anti-ICOS - PE-Cy7 BioLegend 329805 Anti-CTLA4 - PEBioLegend 106305

AB101 treatment significantly reduces A549 and H1975 tumor growthcompared to isotype control antibody. FIG. 26 and FIG. 27 show tumorvolume plotted for the A549 and H1975, respectively, tumors over 30days. Arrows indicate injections with antibody treatments. Each pointrepresents the mean measurement from 7 mice. Error bars denote standarderror of the mean (SEM). Statistical significance was calculated usingMann-Whitney test.

In the isotype control, the volume of A549 and H1975 tumors increasedover time. However, in mice that received AB101 treatment, the A549tumor exhibited slower growth compared to isotype control, while H1975tumor showed regression at day 17 and the growth remained steadythereafter. At randomization on D5, the average A549 tumor volumes forisotype control and AB101 were 63.6 mm³ and 63 mm³ respectively, and onD26, the average tumor volume for isotype control was 378 mm³, whereasthe average tumor volume for AB101 was 198 mm³. Similarly, H1975 tumorvolumes on D5 were 57.8 and 34.2 for isotype control and AB101,respectively and on D26, the average tumor volume for isotype controlwas 164.4 mm³, whereas the average tumor volume for AB101 was 57.4 mm³.

AB101 treatment significantly reduced tumor size of both A549 and H1975tumors. Tumors were excised on D26 and weighed. AB101 reduced the sizeof A549 tumor by 49% relative to the isotype control (average tumorweight: 538.2 mg for isotype control and 273.0 mg for AB101, p=0.003)and H1975 tumor by 60% (average tumor weight: 217.2 mg for isotypecontrol and 85.6 mg for AB101, p=0.0009).

AB101 treatment significantly increased the proportions of CD8⁺ T cellsand myeloid cells amongst the total live cells in the spleen. Theaverage percent CD8⁺ T cells was increased by 1.3 for isotype control to3.3 for AB101 and significantly increased the average percent CD11b⁺cells from 2.1 for isotype control to 4 for AB101.

AB101 treatment also significantly enhanced expression of activationmarkers on human CD8⁺ T cells in spleen. The average MFI for ICOSexpression on CD8⁺ T cells was increased from 318 for isotype control to841 for AB101 and average MFI for OX40 expression was increased from 586for isotype control to 1561 for AB101.

Example 17—AB101 Relieves M2c Mediated Immune Suppression on T Cell inM2c/T Cell Coculture Assay

To evaluate if AB101 can modulate the cancer mediated immune evasion inthe TME, human PBMC-derived T cells were cultured with autologousimmunosuppressive M2c macrophages. AB101 immunomodulatory activities torescue anti-CD3 (OKT3) activated T cells from M2c-mediated immunesuppression were assessed under three treatment regimens, with T cellproliferation and IL-2 production as read outs for treatment efficacy.FIG. 28 shows the experimental design.

To determine if AB101 interferes with the generation of M2-like tumorassociated macrophages, M0 macrophages were polarized to M2c macrophagesin the presence of AB101 or isotype control (“Pre” regimen). Treatmentantibodies were washed out before coculture with T cells. To evaluate ifAB101 treatment rescues T cells from M2c-mediated immune suppression, Tcells were activated with anti-CD3 in the presence of M2c macrophagesand AB101, or isotype control during M2c/T cell coculture (“Post”regimen). To mimic in vivo immunotherapy, Pre and Post regimens werecombined (Pre/Post).

In the first set of experiments, the effect of AB101 on M2c polarizationwas evaluated with human monocyte derived macrophages and T cells fromthree healthy subjects. CD4⁺ and CD8⁺ T cells were activated with OKT3in the presence of autologous M2c macrophages treated with AB101 orisotype control. OKT3 stimulated T cells cocultured with M2c macrophagesalone was used to assess M2c mediated immune suppression. T cellcoculture with IFN-γ+LPS polarized M1 macrophages provided a measure foroptimal T cell activation. M2c/T cell coculture without OKT3 activationresembled resting T cells. FIG. 29 shows that AB101 treatmentsignificantly enhanced the proliferation of CD4⁺ and CD8⁺ T cells overisotype control from 7 to 54% (p<0.01) and from 21 to 83% (p<0.05) ofdividing cells, respectively. M1 macrophages and AB101 treated M2cmacrophages induced similar levels of proliferation. In addition, FIG.30 shows that AB101 Pre-treatment of M2c macrophages significantlyincreased IL-2 production by activated T cells from all three studysubjects, when compared to IL-2 secretion by activated T cells fromAB101-treated or naïve M2c groups. IL-2 levels from coculture with AB101treated M2c macrophages were similar or higher than achieved incoculture with M1 macrophages. As expected, T cells cocultured with M2cwithout OKT3 activation did not produce detectable levels of IL-2.

Next, the effects of AB101 treatment on CD8⁺ T cell/M2c cocultures underPre-, Pre/Post- and Post-regimens were evaluated. The experiment wasperformed with PBMCs from three healthy subjects. CD3⁺ T cells from 3study subjects were activated with anti-CD3 (OKT3, 0.25 μg/mL) in thepresence of M2c macrophages. M2c macrophages were treated with AB101 (20μg/mL), human IgG1 isotype control (20 μg/mL) or media alone duringpolarization (Pre, before coculture). T cells were harvested 72 h afteranti-CD3 stimulation and proliferation was quantified by flow cytometry.P values were calculated by Dunnett's T3 multiple comparisons test forM2c, AB101 and IgG1 isotype control treatment groups (p<0.05, *; p<0.01,**p<0.001, ***).

FIG. 31 shows that AB101 treatment significantly enhanced CD8⁺ T cellproliferation under Pre- and Post-regimens, when compared to the isotypecontrol group (p<0.05). AB101 pre- and post-regimens increased thepercent of divided CD8⁺ T cells from 23 to 42% and from 26 to 47%,respectively when compared to the corresponding isotype control groupvalues. The largest increase in CD8⁺ T cell proliferation was observedin AB101 Pre/Post group, with 49% divided CD8⁺ T cells, compared to the22% dividing CD8⁺ T cells of the isotype control Pre/Post control group(p=0.062).

FIG. 32 shows the corresponding IL-2 data for the individual studysubjects. All three subjects in the AB101 treatment groups significantlyincreased the IL-2 production of CD8⁺ T cells when compared to M2c aloneand isotype control groups. The highest IL-2 secretion was achieved withthe Pre/Post combination treatment of M2c/T cell cocultures. Theproliferation and IL-2 data of the three AB101 treatment regimensindicated that AB101 not only affected the polarization of M2c cell butalso mitigated the M2c mediated immune suppression during T cell/M2ccoculture.

FIG. 33 and FIG. 34 show the compiled proliferation data for Pre andPre/Post AB101 treatment. AB101 had a greater effect on theproliferation of CD8⁺ T cells than on CD4⁺ T cell proliferation. AB101treatment significantly enhanced the proliferation of CD8⁺ T cells overthe corresponding isotype control values under Pre (n=16, p<0.001) andPre/Post-regimens (n=13, p<0.001), respectively. CD4⁺ T cellsproliferated in response to AB101 Pre- and Pre/Post-regimens with anincrease in percent of divided cells from 31 to 44% (Pre, n=18, p<0.01)and 42 to 49% (Pre/Post, n=14, p<0.05) when compared to isotype treatedM2c macrophages.

FIG. 35 shows that treatment with AB101 during polarization of M2c cellsalso significantly enhanced IL-2 production of T cells in the M2c/T cellcoculture assay. T cells in the Pre-treatment AB101 group producedsignificantly higher levels of IL-2, with a median of 292 ng/mL, almostthree-fold higher than the 104 ng/mL from T cells in the correspondingisotype control treatment group (n=21, p<0.0001). The AB101 Pre/Posttreatment generated an IL-2 response by T cells of 333 ng/mL, 50%greater than the 227 ng/mL from isotype control treated M2c/T cellcocultures; this difference, however, did not reach significance.

Example 18—AB101 is More Potent than AB104 in Restoring T CellProliferation and Cytokine Response by T Cells Cocultured with M2cMacrophages

To determine if AB101 mediated relief of immune suppression requiresinteractions with Fc receptors expressed by M2c macrophages, weevaluated the AB101-IgG4 (AB104) and AB101-IgG1Fcnull (AB102) isotypesin the M2c/T cell coculture assay. The AB101 IgG1-Fc region binds toCD64, CD32 and CD16 on M2c macrophages, whereas the binding of theIgG1Fcnull isotype to Fcγ receptors is minimal. Like the IgG1 Fc region,the IgG4 Fc region has a nanomolar affinity to CD64 but does not usuallybind to CD32 or CD16 Fc receptors. The ability of AB101, AB102 and AB104isotypes to relieve M2c mediated immune suppression on T cellproliferation was compared with cells from 5 study subjects.

T cells isolated from 4 (CD8⁺ T cells) and 5 (CD4⁺ T cells) humansubjects were activated with anti-CD3 (OKT3, 0.25 μg/mL) in the presenceof M2c macrophages. M2c/T cell cocultures were treated underPre/Post-regimens with 20 μg/mL of the indicated isotypes of AB101. M2/Tcell coculture alone was used as a control for M2c mediated immunesuppression. T cells were harvested 72 h after anti-CD3 stimulation andproliferation was quantified by flow cytometry. Symbols representindividual study subjects. P values were calculated by paired, twotailed t-test comparing the indicated treatment groups (p<0.05, *; ns,not significant).

FIG. 36 shows that AB101 Pre/Post-regimen significantly (p<0.05)enhanced the mean proliferation of anti-CD3 activated CD8⁺ and CD4⁺ Tcells, when compared to IgG1 isotype control (52 to 78% CD8⁺ T cells; 46to 65% CD4⁺ T cells) and to AB102 (52 to 78% CD8⁺ T cells; 42 to 65%CD4⁺ T cells). AB104 and AB102 treatment only marginally enhanced theproliferative response of T cells when compared to the respectiveisotype controls. Only the proliferative response of CD4⁺ T cells in theAB104 treatment group reached significance over the IgG4 isotype controlgroup (39 to 47% divided cells, p<0.05).

FIG. 37 shows that AB101 Pre/Post-regimen had a strong and significantstimulatory effect on OKT3 mediated CD8⁺ T cell proliferation (70%divided cells) when compared to IgG1 isotype control (40% divided cells,p<0.05) and AB104 treatment (41% divided cells, p<0.05). Additionally,AB101 Post-regimen demonstrated significantly enhanced CD8⁺ T cellproliferation (54% divided cells), when compared to isotype control (27%divided cells, p<0.05), or AB104 treatment (20% divided cells, p<0.05).AB104 treatment did not significantly improve the proliferative responseover the IgG4 isotype control in Pre/Post- or Post-regimen.

AB101 treatment during M2c/T cell coculture (Post-regimen) relieved M2cmediated immunosuppression and induced a potent cytokine response byanti-CD3 activated CD8⁺ T cells. CD8⁺ T cells isolated from 3 studysubjects were activated with anti-CD3 (OKT3, 0.25 μg/mL) in the presenceof M2c macrophages. M2c/T cell cocultures were treated underPost-regimen with 20 μg/mL of AB101, human IgG1 isotype control, AB104,human IgG4 isotype control, and media alone (M2c). Supernatants weretaken 72 h after anti-CD3 stimulation and cytokine secretion wasquantified by magnetic bead-based immunoassay. P values were calculatedby paired, two tailed t-tests comparing the indicated treatment groups(p<0.05, *; p<0.01, **; p<0.001, ***; ns, not significant).

FIG. 38 shows that AB101 significantly enhanced the IFN-γ and perforinlevels in all study subjects when compared to the IgG1 isotype control.Table 6 shows that the mean IFN-γ, perforin and IL-6 levels in the AB101treatment groups increased from 530 to 1600 μg/mL (IFN-γ), 210 to 1900μg/mL (perforin) and 203 to 690 μg/mL (IL-6) in comparison to IgG1isotype control values. In addition, AB101 treatment restored TNF-αsecretion in 2 of the 3 study subjects with significant increase overthe corresponding IgG1 isotype control values from 60 to 830 μg/mL andfrom 1 to 120 μg/mL. As shown in Table 1 the AB101 Pre/Post-regimenconfirmed the results observed with the Post-regimen group by inducingsimilar cytokine levels for perforin and tested cytokines. AB104 did notrelieve M2c mediated immune suppression in any of the treatment groups.The IL-10 levels in the evaluated M2c cocultures were at the lowerdetection limit of the assay under all treatment conditions.

TABLE 6 AB101 Rescued CD8⁺ T cell Cytokine Response from M2c MacrophageMediated Immune Suppression Cytokine Secretion by CD8⁺ T cells [pg/mL]Controls Pre/Post-Regimen Post-Regimen No antibody AB101 AB104 AB101AB104 Cytokines M1 M2 Ab Iso Ab Iso Ab Iso Ab Iso IL-6 Mean  243  27 744  62 12  51  686 203 22  77 SEM  113  16  222  36  5  28  201  86 21 53 IL10 Mean   4  20  26  11  9  13  20  16 10  13 SEM   4  10  17  0 5  3  10  3  7  3 IFN-γ Mean 3016 259 1730 461 14 134 1615 531 17 220SEM 1209 295 1441 549  5 153 1439 632  2 255 TNF-α Mean  53  14  330  23 2  12  312  31  2  14 SEM  24  15  310  25  1  12  272  32  2  15Perforin Mean  220  91 2459 144 57  72 1912 213 48  73 SEM  129  34 1945124  8  24 1494 147 19  23

The corresponding cytokine and perforin results for AB101 Post- andPre/Post-regimen M2c/CD4⁺ T cell cocultures are shown in FIG. 39 andTable 2. CD4⁺ T cells isolated from 3 healthy study subjects wereactivated with anti-CD3 (OKT3, 0.25 μg/mL) in the presence of M2cmacrophages. M2c/T cell cocultures were treated under the Post regimenswith AB101 (20 μg/mL), human IgG1 isotype control (20 μg/mL), AB104 (20μg/mL), human IgG4 isotype control (20 μg/mL), and media alone (M2c).Supernatants were taken 72 h after anti-CD3 stimulation and cytokinesecretion was quantified by magnetic bead-based immunoassay. P valueswere calculated by paired, two tailed t-tests comparing the indicatedtreatment groups (p<0.05, *; p<0.01, **; ns: not significant).

AB101 significantly enhanced the IFN-γ, TNF-α and perforin levels in allstudy subjects when compared to the IgG1 isotype control and AB104. Themean IFN-γ, TNF-α, perforin and IL-6 levels in the AB101 treatmentgroups increased from 770 to 1700 μg/mL (IFN-γ), 420 to 1400 μg/mL(TNF-α), 220 to 780 pf/mL (perforin) and 1300 to 5100 (IL-6) μg/mL incomparison to IgG 1 isotype control values. AB101 Pre/Post-regimenconfirmed the results observed with the Post-regimen group by inducingsimilar cytokine levels for perforin and tested cytokines. AB104 did notenhance cytokine or perforin responses when compared to thecorresponding levels of the IgG4-isotype control group.

TABLE 7 AB101 Rescued CD4⁺ T cell Cytokine Response from M2c MacrophageMediated Immune Suppression Cytokine Secretion by CD4⁺ T cells [pg/mL]Controls Pre/Post-Regimen Post-Regimen No antibody AB101 AB104 AB101AB104 Cytokines M1 M2 Ab Iso Ab Iso Ab Iso Ab Iso IL-6 Mean 372 112 50391092 1015 940 5129 1330 1101 738 SEM 205 33 1813 236 214 362 2372 388380 153 IL10 Mean 93 67 576 232 171 132 533 218 164 129 SEM 50 35 171106 53 35 103 106 68 67 IFN-γ Mean 4202 84 1609 721 562 416 1678 774 588366 SEM 1012 53 251 291 176 96 195 345 255 194 TNF-α Mean 175 133 1356124 427 500 1377 423 417 419 SEM 95 82 402 70 70 95 261 59 87 121Perforin Mean 117 64 685 173 122 99 776 224 138 94 SEM 58 5 195 50 33 9231 103 48 34

In conclusion, the rescue of the T cell cytokine response by AB101 fromM2c mediated immune suppression and the lack of efficacy by the AB104isotype suggest that AB101 Fc receptor interactions may be required forthe AB101 function.

Example 19—AB101 Treatment Enhanced the Cytotoxic Activity of CD8⁺ TCells

To determine if AB101 enhances tumor cell killing by CD8⁺ T cells in theTME, the cytotoxic activity of CD8⁺ T cells stimulated with anti-CD3antibody in the presence of M2c macrophages was evaluated. In thisstudy, the tumor antigen specific, T cell mediated, killing of tumorcells was substituted with Bispecific T cell Engager (BiTE®) technology.BiTE antibodies are fusion proteins, consisting of variable domains oftwo monoclonal antibodies, that are designed to bridge cancer cells toCTLs. One variable domain targets an antigen on the cancer cell surface,and the other variable domain engages CD3 on the surface of a T cell.Upon binding of both arms of the BiTE® antibody, T cells and cancercells are forced within proximity of one another. As a result, acytolytic synapse is created between the T cell and cancer cell,perforin and granzymes are released from the T cell, and tumor celldeath occurs.

The CD19-CD3 BiTE antibody was used to assess the efficacy of AB101 inT-cell-mediated tumor cell killing of Raji B cell lymphoma cells whichexpress the CD19 target of BiTE. AB101 treatment of the M2c/CD8⁺ T cellcoculture relieves M2c mediated immune suppression and may expand andenhance the cytolytic activity of CD8⁺ T cells. CD8⁺ T cells may alsokill Raji cells by allogenic HLA-restricted cytolysis in the absence ofBiTE. K562 cells (chronic myeloid leukemia cell line) that do notexpress the BiTE or HLA were included for the evaluation ofnon-HLA-restricted cell death by CD8⁺ T cells.

A coculture of M2c macrophages and autologous human primary CD8⁺ T cellswas activated with anti-CD3 and expanded for 3 days. Cocultures weretreated with AB101 (20 μg/mL), human IgG1 isotype control (20 μg/mL) ormedia alone under Pre-, Pre/Post-, and Post-regimen. Under Pre- andPost-regimen, therapeutic antibodies were added only during M2cpolarization and during M2c T cell coculture, respectively. P valueswere calculated by paired, two tailed t-test comparing AB101 to M2c andisotype control treatment groups, respectively (p<0.05, *; p<0.01, **:p<0.001, ***; p<0.0001, ****; ns: not significant). The workflow of theM2/T cell coculture assay with cytotoxicity readout is shown in FIG. 28.

FIG. 40 shows that AB101 treatment during polarization of M0 to M2macrophages or during M2c/T cell coculture enhanced the killing of Rajicells in the presence of the CD19-CD3 BiTE® antibody significantly, whencompared to the isotype control. The percent of Raji cell deathincreased from 66 to 77% (p<0.01) under Pre, from 64 to 83% (p<0.001)under Pre/Post and from 65 to 82% (p<0.01) under Post-regimens. The RajiBiTE® killing assay had a small dynamic range and high background withresting CD8⁺ T cell (grey bar, black circles) cultured with M2c cellskilling 71% of Raji cells.

Notably, AB101 treatment also increased the cytotoxic activity ofnon-HLA-restricted CD8⁺ T cells targeting K562 cancer cells. Pre/Post-and Post AB101 treatment of M2c macrophages and T cells enhanced thetumor cell killing from 12 to 41% under Pre/Post (p<0.01) and from 13 to36% (p<0.05) under Post conditions compares to the related isotypecontrol values.

Next, the effect of AB101 on the cytotoxic activity of CD8⁺ T cells froma panel of human subjects was evaluated. CD8⁺ T cells from 8 studysubjects were propagated in the presence of autologous M2c macrophageswith anti-CD3 (OKT3, 0.25 μg/mL). M2c macrophages alone and cocultureswere treated with AB101 (20 μg/mL), human IgG1 isotype control (20μg/mL) or media alone under Pre-, Pre/Post-, and Post-regimens. T cellswere harvested 72 h after anti-CD3 stimulation and cultured with Rajicells in the presence or absence of CD19-CD3 BiTE antibody. Cell deathof Raji cells was determined by flow cytometry 18 h after cytolysisassay setup. P values were calculated by paired, two tailed t-testcomparing AB101 to M2c and human IgG1 isotype control treatment groups,respectively (p<0.01, **; p<0.001, ***).

As shown in FIG. 41 , AB101 Pre/Post treatment significantly increasedthe CD8⁺ T cell-mediated BiTE®-assisted Raji cell killing from 49.5 to63.5% (p<0.001) when compared to the human IgG1 isotype control group.Without BiTE, CD8⁺ T cells from the AB101 group enhanced the cytolysisof Raji cells from 35 to 43% (p<0.01).

Next, the effect of AB101 on the cytotoxic activity of non-HLArestricted CD8⁺ T cells was evaluated. CD8⁺ T cells from 8 studysubjects were propagated in the presence of autologous M2c macrophageswith anti-CD3 (OKT3, 0.25 μg/mL). M2c macrophages alone and cocultureswere treated with AB101 (20 μg/mL), human IgG1 isotype control (20μg/mL), or media alone under Pre-, Pre/Post-, and Post-regimens. T cellswere harvested 72 h after anti-CD3 stimulation and cultured with K562cells. Cell death of K562 cells was determined by flow cytometry 18 hafter assay setup. P values were calculated by paired, two tailed t-testcomparing AB101 to M2c and human IgG1 isotype control treatment groups,respectively (p<0.01, **).

FIG. 42 shows that AB101 treatment had the strongest effect on cytolyticCD8⁺ T cells activity in the K562 killing assay. The mean cell death inthe AB101 treatment group was twice as high as a the human IgG1 isotypecontrol group, increasing the mean K562 killing from 14 to 29%,respectively (p<0.01).

Example 20—AB101 Modulates the Expression of Chemokine Receptors on TCells Cocultured with M2c Macrophages

To determine if AB101 alters the activation state of CD4⁺ and CD8⁺ Tcells, the expression of chemokine receptors and markers of activationor exhaustion were evaluated in the M2c/T cell coculture assay as shownin FIG. 28 .

Anti-CD3 activated T cells were cocultured with autologous M2c cellstreated with AB101 or isotype control during polarization. Anti-CD3activated T cells cocultured with naïve M2c or IFN-γ LPS activated M1macrophages were included as controls for immune suppression and immuneactivation, respectively. Resting T cells were cocultured with M2cmacrophages without anti-CD3 activation. Three days after anti-CD3activation, T cells were analyzed by flow cytometry. The flow cytometrystaining panel 1 (Table 8) included antibodies for activation markers(CD25 and CD69), resting T cell marker CD127, as well as chemokinereceptors CXCR3, CXCR4 (CD194) and CCR6 (CD196) typically used todifferentiate CD4⁺ T cell subsets. Flow cytometry antibody panel 2(Table 8) contained the T cell activation and exhaustion markers LAG3,OX40, PD-1, ICOS and CTLA-4.

T cells isolated from 3 study subjects were activated with anti-CD3(OKT3, 0.25 μg/mL) in the presence of M2c macrophages. M2c/T cellcocultures were treated during M2c polarization with AB101 (20 μg/mL),human IgG1 isotype control (20 μg/mL), or media alone. M1/T cellcoculture was included as a positive control. Supernatants wereharvested 72 h after anti-CD3 stimulation for flow cytometry. Heatmaprepresents the FlowSOM cluster analysis of the combined treatment groupsof all study subjects.

The phenotype of CD4⁺ T cells was evaluated using flow cytometry panel 1and the FlowJo FlowSOM plugin for non-biased clustering. As shown inFIG. 43 , FlowSOM clustering of CD4⁺ T cells from all M2c/T cellscoculture groups identified 8 clusters (numbered 0-7) with differentialexpression levels for the surface markers. Cluster 1 represents restingT cells and cluster 6 resembles activated Th1-like T cells.

TABLE 8 Antibody Panels Used to Assess Expression of Chemokine Receptorson CD4⁺ T cell Subtypes and Exhaustion/Activation Markers Antibody Panel1: Antibody Panel 2: T Cell Phenotyping Panel Exhaustion/ActivationSurface CD4 - FITC CD4 - FITC markers CD69 - PE-Cy7 CD8 - APC CD25 - APCLAG3 - BV421 CD127 - PE OX40 - BV510 CXCR3 - PerCP-Cy5.5 PD-1-PerCP-Cy5.5 CD194 (CCR4) - BV421 ICOS - PE-Cy7 CD196 (CCR6) - BV510CTLA4 - PE

A visualized representative example of how AB101 treatment influencedthe proportions of CD4⁺ T cells is shown in Table 9 and FIG. 43 . Themajority (mean=74%) of T cells cocultured with M2c cells withoutanti-CD3 activation were found in cluster 1 (from 3 out of 3 studysubjects). Cluster 1 cells had a resting phenotype with low or no CD69,CXCR3, CCR4 or CD25 expression, and elevated expression of CD127. In thepresence of the immune-activating M1 polarized macrophages, the majorityof T cells (57%, mean of all 3 subjects) adopt an activated phenotypecharacterized by high expression of CXCR3 and low expression of CCR4,CD127, CCR6, and activation markers CD25 and CD69 (cluster 2). M1coculture also induces a unique smaller subset with elevated expressionof CD25, CXCR3 and CCR4 (cluster 7, 21% of T cells cocultured with M1).M2c cells had immunosuppressive effects on anti-CD3 activated CD4⁺ Tcells in coculture with 46% of the T cells in the resting phenotypecluster 1. In addition, 32% of the T cells of the M2c alone group werefound in the activated T cell phenotype cluster 2.

TABLE 9 AB101 Modulates the Distribution of Activated CD4⁺ T cells inFlowSOM Cluster Distribution of CD4⁺ T cells in FlowSOM clusters [%]Resting Isotype T cells control AB101 FlowSOM M2c M2c + OKT3 M2c + OKT3M2c + OKT3 M1 + OKT3 Cluster Mean SEM Mean SEM Mean SEM Mean SEM MeanSEM 0 1.7 0.3 3.3 0.9 3.0 0.7 5.9 1.6 2.9 0.8 1 73.1 7.8 46.3 2.9 51.31.6 12.3 7.3 8.9 3.7 2 19.8 5.7 31.9 5.3 27.4 1.9 40.5 9.8 55.6 6.1 31.3 0.8 4.3 1.5 4.2 0.7 14.0 4.5 1.1 0.5 4 2.9 0.8 9.6 1.7 8.7 1.5 16.03.0 3.3 0.5 5 0.2 0.0 0.3 0.1 0.2 0.1 0.6 0.1 3.9 1.3 6 0.4 0.2 3.3 0.54.4 1.8 9.3 1.0 3.4 0.8 7 0.7 0.3 1.0 0.1 0.9 0.1 1.4 0.1 20.8 1.5

FIG. 43 shows that Anti-CD3 activated CD4⁺ T cells from the isotypecontrol treatment group had a similar distribution profile to the Tcells from the corresponding M2c alone group, with a mean of 51% of theT cells in cluster 1 and 27% of T cells in cluster 2 (Table 9).

In contrast, treatment with the AB101 alleviated the suppressive effectsof the M2c polarized macrophages. AB101 significantly enhanced theproportion of T cells sharing the activated phenotype of cluster 2compared to isotype control isotype control from 27 to 40% (p<0.05). Inaddition, AB101 significantly decreased the proportion of cells sharingthe phenotype of resting cells from 51 to 13% (cluster 1; p<0.0001).This distribution resembled the phenotype patterns of T cells which havebeen stimulated in the presence of M1 macrophages.

Clusters 3, 4 and 6 were also elevated by treatment with AB101 whencompared to isotype control group. As shown in FIG. 43 , the differencesin the 3 clusters reached significance in 2 out of the 3 evaluatedsubjects (p<0.0001) related to the respective M2c and isotype controls.

Cluster 3 and 4 are defined by high expression of CXCR3, mid expressionof CCR4 and presence of CD127 with high (Cl. 3) or low (Cl. 4)expression of the activation marker CD69. The phenotype of cluster 3 isnot shared with either M1 or resting T cells, appearing unique to AB101treatment.

Cluster 6 represents the phenotype of activated Th1-like T cells withhigh expression of CXCR3 and CD69 and minimal expression of CCR4 andCCR6. AB101 treatment increased the mean percentage of proportion of Tcells in cluster 6 from 4.4% of the isotype treatment group to 9.3%(Table 9).

In conclusion, the FlowSOM analysis of the CD4⁺ T cell phenotypesexpanded by M2c coculture indicate that AB101 treatment relieves M2cmediated immune suppression and induces the expression of unique T cellphenotypes highlighted by the expression of CXCR3 and CCR4. AB101treatment also increased the proportion of activated Th1-like CXCR3⁺ Tcells in the M2c cocultures.

To further investigate the ability of AB101 to block M2c-mediatedsuppression resulting in enhanced activation of T cells, CD4⁺ and CD8⁺ Tcells were assessed for the markers of activation and exhaustion LAG3,OX40, PD-1, ICOS and CTLA-4. Clustering of CD4⁺ and CD8⁺ T cells byFlowSOM identified 5 CD4⁺ (numbered 0-4) and 4 CD8⁺ (numbered 0-3)clusters.

The low expression of LAG3, OX40, PD-1 and CTLA-4 in Cluster 0 (CD4⁺ Tcells) and Cluster 3 (CD8⁺ T cells) are indicative of a restingphenotype. The increased expression of PD-1 and ICOS are in cluster 1(CD4⁺ T cells) and cluster 3 (CD8⁺ T cells) represents the activatedphenotype in this study (FIG. 44 ).

Cluster 0 (ICOS⁺ PD-1⁻ LAG3⁻ CTLA4⁻ OX40⁻) represents 90% ofun-stimulated CD4⁺ T cells and cluster 3 (ICOS^(lo) PD-1⁻ LAG3⁻ CTLA4⁻OX40⁻) represent 93% of resting CD8⁺ T cells cultured with M2cmacrophages without anti-CD3 activation (FIG. 44 ). When cocultured withM2c polarized macrophages or treated with isotype control (anti-CD3activated CD4⁺ and CD8⁺ T cells primarily fall into the correspondingresting cluster 0 (65% of CD4⁺ T cells are) and cluster 3 (64% of CD8⁺ Tcells) confirming M2c cell mediated immune suppression.

AB101 significantly enhances the activation of CD4⁺ and CD8⁺ T cellswhen compared to the M2c alone or the isotype control treatment groups.Eighty-two percent of CD4⁺ T cells and 93% of CD8⁺ T cells of the AB101treatment group are found in the respective activated T cell phenotypecluster 1 (ICOS^(hi) PD-1⁺ LAG3⁻ CTLA4⁻ OX40^(lo)) and cluster 0(ICOS^(hi) PD-1⁺ LAG3^(lo) CTLA4⁻ OX40^(lo)) shared with T cellscocultured with M1 polarized macrophages.

Example 21—AB101 Modulated the Expression of m2c Surface Markers

AB101 treatment during polarization of M0 to M2c macrophages rescuedanti-CD3 activated T cells from M2c-mediated immune suppression in theM2c/T cell coculture assay. To determine if AB101 modulates theexpression of surface markers and immune checkpoints on M2c, 5-day oldM0 macrophages were polarized with IL-10 to M2c macrophages in thepresence of AB101 or isotype control (20 μg/mL) and then stained with apanel of macrophage phenotyping antibodies. The flow cytometry profileswere compared to naïve, untreated M2c cells and LPS+IFN-γ polarized M1macrophages. M2c macrophages express the M2c markers CD163, CD206 andMer-TK, the Fcγ receptors CD16, CD32, CD64, the pattern recognitionreceptor TLR2, the TNFR family member CD40. As expected, after IFN-γtreatment, M1 macrophages expressed higher levels of HLA-Class II andthe checkpoint ligand PD-L1 when compared to M2c macrophages. Incontrast, M2c macrophages showed higher levels of the immune suppressiveligands Siglec-15 and LILRB2 than M1 macrophages. The evaluated surfacemarkers, costimulatory molecules, and receptors CD86, CD91, CD150,Calreticulin, Dectin-1, TIM4 and TLR4 are not expressed on M2c cells.

To further analyze the CXCR3 expression by activated CD4⁺ T cells theFlowSOM clusters 3 to 6 were combined based on their CXCR3⁺ CD69⁺ CD25⁺T cell phenotype. The proportions of the resulting phenotypes are shownas pie charts in FIG. 45 and represent the phenotypes in the table inFIG. 45 .

AB101 treatment increased the proportion of activated CXCR3⁺, CD4⁺ Tcells expressing the activation markers CD69 and CD25 from 18% to 40%when compared to the isotype treatment group. The OR2572 and M2c alonetreatment groups had comparable distribution profiles.

To quantify the modulatory effect of AB101 on surface marker expression,the mMFI values of the phenotyping antibodies on AB101 treated M2c cellswere normalized to the corresponding markers on isotype control(nMFIiso) or untreated, naïve M2c macrophages (nMFIM2c) from up to 10study subjects (FIG. 46 ). AB101 induced a highly significant reductionof CD16 (34% nMFIiso, p<0.0001) and CD64 (30% nMFIiso, p<0.0001) as wellas a significant decrease of TLR2 (66% nMFIiso, p<0.05) relative toisotype control treated M2c cells. Similar trends were observed whenAB101 M2c surface marker MFIs were normalized to naïve M2c macrophages.The nMFiM2c of surface markers from AB101 treated macrophages werereduced significantly for CD16 (44% nMFIM2c, p<0.001), CD64 (30%nMFIM2c, p<0.0001), TLR2 (63% nMFIM2c, p<0.05) and Siglec-15 (66%nMFIM2c, p<0.05). AB101 treatment enhanced the expression of HLA-ClassII and did not significantly affect the expression of CD163, CD206,MerkTK, LILRB2 and PD-L1, when compared to the M2c macrophage controls.

In summary, AB101 treatment during polarization of M2c macrophagesreduced the expression of the innate receptor TLR2 and the checkpointligand Siglec-15. In addition, it inhibited IL-10 induced upregulationof CD16 and CD64 on M2c cells.

Example 22—AB101 Binding Increased Protection (Slower HD Exchange) ofRegions in SRCR Domain 3 and 4 and Exposed (Faster HD Exchange) Regionsin Domain 2, 5, and 9 of CD163

A comparative HDX study was conducted to examine the effects of IgGbinding on the CD163. Due to the large number of peptides derived fromthe excess Ab present in the complex many resulted in many peptidesoverlapping in m/z and retention times. Ultimately, after filtering outnoisy and overlapped peptides a final set of 107 peptides in the pepsindata, corresponding to a coverage of 74% with an average redundancy of1.28. For the Nepenthesin II data set the final filtered peptide countwas 230 corresponding to 87% coverage with an average redundancy of 2.8.When combining the data sets with both proteases the total sequencecoverage is 93% with an average redundancy of 4.1.

Hydrogen/Deuterium Exchange with Mass Spectrometry: Starting stocksolutions of recombinant human CD163 (rhCD163) was diluted to 0.36 mg/mLin phosphate buffered saline (PBS) pH 7.2. Internal exchange reporters:tetrapeptide PPPF (SEQ ID NO: 27) or tripeptide YPI were added to eachsolution for a final concentration of 1 μM. The antibody-complexedsample was made the same way but included 1.38 mg/mL of AB101, whichcorresponds to a 3-fold molar excess over rhCD163. These workingsolutions were incubated at 22° C. and stored at 4° C. for 1 day priorto starting the deuterium exchange reactions. 104 of the working proteinsolution was diluted 10-fold into 90 μL deuterated HBS buffer: (20 mMHEPES, pH 7.2, 150 mM NaCl, 2 mM CaCl₂), 95% D₂O) and incubated at 22°C. for 3 sec, 15 sec, 1 min, 5 min, 30 min, 4 hrs, or 20 hrs. Thedeuterated HBS also contained 0.2 μg/mL of bradykinin to provide a fullydeuterated reference compound in all experiments in order to monitorback-exchange. An additional sample was incubated at 37° C. for 20 hoursas a highly deuterated sample. Exchanged samples were added to an equalvolume (100 μL) of ice-cold quench buffer: 1M TCEP, 0.2% formic acid(FA), for a final pH of 2.5. Samples were flash frozen in an ethanol-dryice bath (−60° C.) and subsequently stored at −80° C. until LC-MSanalysis. Undeuterated reference samples were prepared identicallyexcept diluted into aqueous HBS buffer.

Sample processing with immobilized pepsin protease: Frozen samples werethawed on a 5° C. block for 4 minutes prior to injection onto a loadingloop. The loaded sample was passed over a custom packed pepsin column(Porcine pepsin immobilized on POROS 20-AL resin; 2.1×50 mm column) keptat 12° C. with a flow of 0.1% trifluoroacetic acid (TFA) and 2%acetonitrile (ACN) at 200 μL/min. Digested peptic fragments were trappedonto a Waters XSelect CSH C18 XP VanGuard Cartridge (2.1×5 mm, 2.5 μm).After 5 minutes of loading, digestion, and trapping, peptides wereresolved on an analytical column (Waters CSH 1×100 mm, 1.7 μm, 130 Å)using a gradient of 3% to 40% solvent B for 9 minutes (A: 0.1% FA,0.025% TFA, 2% ACN; B) 0.1% FA in ACN). The LC system was coupled to aWaters Synapt G2-Si performing full scans over the m/z range of 300-2000with ion mobility separation enabled. The source conditions wereoptimized to minimize loss of deuterium during desolvation. Undeuteratedsamples were run prior to and at the end of all the LC-MS runs. Duringthe analytical separation step, a series of 2504 injections were used toclean the pepsin column: 1) 0.1% Fos-12 with 0.1% TFA; 2) 2 M GndHCl in0.1% TFA; 3) 10% acetic acid, 10% acetonitrile, 5% isopropanol. Aftereach gradient the trapping column was washed with a series of 2504injections: 1) 10% FA; 2) 30% trifluoroethanol; 3) 80% methanol; 4) 66%isopropanol, 34% ACN; 5) 80% ACN. During the trap washes the analyticalcolumn was cleaned with three rapid gradients. These cleaning steps werenecessary to ensure that the level of carry-over was below 5% for eachpeptide analyzed.

Sample processing with immobilized Nepenthesin II protease: Sampleprocessing described above was repeated except Nepenthesin II proteaseimmobilized on POROS 20-AL was used for the online digestion step.

To first ensure that the exchange and sampling conditions were identicalfor the unbound and antibody-bound states we first examined the exchangeprofile of the internal standard peptides. For both data sets the PPPI(SEQ ID NO: 28) and YPI standards were consistent between the unligandedand antibody-bound data sets ensuring that the conditions wereconsistent and even subtle changes can be interpreted to be due toaltered protein structure and/or dynamics. Furthermore, the fullydeuterated bradykinin peptide that was incorporated into the deuteratedbuffer showed identical deuterium levels, ensuring that the level ofback-exchange was not variant among samples within each data set.

The comparison of the HDX kinetics between the antibody-bound unboundstates was used to assess the changes throughout the antigen in responseto antibody binding. A summary of the changes for the pepsin andNepenthesin II data sets are shown in FIGS. 50 and 51 . The changes arecolored on the primary sequence by whether there were small (smallchanges at a single time point) or large changes (beyond two standarddeviations or seen at several time points) in HDX kinetics in responseto antibody binding. We note that for these comparisons we only utilizedchanges that were statistically significant as assessed by the standarddeviation among the replicates, along with making sure that alloverlapping peptides covering nearly the same region are in agreement.By these criteria we ensure that we make only the most conservativeinferences from the data sets.

In the pepsin data sets shown on FIG. 47 , there were two sites thatshowed increased protection upon antibody binding. One site includes theglycopeptide 271-285, which showed a drastic increase in protection atseveral time points, and several peptides covering the C-terminallyadjacent region 286-299 which also show a significant decrease inexchange kinetics. A second site includes three peptides within residues405-418 which show a slight increase in protection. A large number ofpeptides showed an increase in flexibility (faster exchange) uponantibody binding. The most striking were at residues 125-139 and479-488, while more subtle increases were observed at residues 887-910and 918-933.

The Nepenthesin II data set shown in FIG. 48 overall showed similarchanges to those observed with pepsin. The largest increases inprotection were evident at residues 279-286, with the region justN-terminal to this (271-278) showing a small increase. The residues408-415 also showed a small increase in protection upon antibodybinding. As in the pepsin data, there was an increase in flexibilityacross large sections of the protein. The largest increase inflexibility was observed at residues 471-483, with smaller changesacross many more parts of the protein. The consistency with thesedatasets strongly indicates the epitope of OR2805 lies with withinresidues 279-285 and may also include the neighboring sequence (279-299)as well as residues 405-418. Other regions may also be involved thatwere not observed by HDX-MS.

Much like in the pepsin data, there was also an increase in flexibilityacross large sections of the protein in the Nepenthesin II data set. Thelargest increase in flexibility was observed at residues 471-483, withsmaller changes across many more parts of the protein (FIG. 49 ).Overall, the large changes distal to the proposed epitope are consistentwith a large-scale allosteric effect upon antibody binding. The factthat all of the changes distal to the epitope are becoming more exposedindicate that antibody binding loosens some secondary structure acrossthe protein, likely by influencing the interactions with neighboringprotein domains. The fact that changes are seen at both the veryN-terminal and C-terminal domains indicates that this protein hasseveral inter-domain interactions in its native (unliganded)conformation.

FIG. 49 shows a schematic summary of the effect of AB101 binding tohuman CD163 ECD. In the presence of AB101, regions within domains SRCR 3and 4 were protected from proton-deuterium exchange. Concomitantly withreduced exchange in domains 3 and 4, SRCR domains 2, 5, and 9 displayedincreased exchange indicative of greater exposure to solvent upon AB101binding.

Example 23—AB101 Binds Truncated Human CD163 ECD SRCR Domain 1-5Fragment

The extracellular domain of human CD163 was truncated after SRCR domain5 and retained a C-terminus histidine tag. This CD163 ECD fragment wasexpressed in HEK293-6E cells and purified using IMAC methods, as shownin FIG. 50 . AB101 binds truncated ECD with sub-nanomolar EC50 asindicated in Table 10, however as expected RM3/1 does not bind. RM3/1binding epitope has been mapped to SRCR domains 8 and 9. The truncatedCD163 ECD SRCR 1-5 contains domains 3 and 4 that were identified byHDX-MS to contain the AB101 binding epitope. That AB101 maintainsbinding to SRCR 1-5 further supports the epitope identification resultsfrom the HDX-MS study.

TABLE 10 EC50 values for AB101, RM3/1, EDHu-1, and 215927 binding totruncated CD163 ECD SRCR 1-5. EC50 (nM) Antigen AB101 R11 RM3/1 EDHu-1215927 huCD163 ECD 0.26 0.061 0.052 0.017 huCD163 SRCR 1-5 0.76 — 0.0500.020 Ratio SRCR 1-5/ECD 2.9  — 0.96  1.17 

Example 24—AB101 Binds to Human but not Cynomolgus Recombinant CD163 ECDProtein and a Single Point Mutation, E323K, in Cynomolgus CD163 ECDConfers AB101 Binding on Par with Human CD163 ECD

In EXAMPLE 6, using an ELISA assay with recombinant CD163 proteinsimmobilized to plastic, the AB101 binding to recombinant human wasdetermined from 14-point dose-response curves in 26 separate experimentsto have a geometric mean EC50 value of 0.52 nM. AB101 did not showbinding to the recombinant murine CD163, while a commercially availablerat anti-muCD163 (Thermo Fisher Scientific #14-1631-82) antibody boundas expected.

As a result of the failure of AB101 to bind murine CD163 and the lowamino acid sequence identity with human CD163 (72.9%), focus wasdirected toward a second species often used in preclinical toxicologystudies, namely non-human primate (NHP) cynomolgus. Cynomolgus CD163shares a 96.5% amino acid sequence identity with human CD163. Productionof human and cynomolgus CD163 ECD with a C-terminal 8 histidine tag (SEQID NO: 22) in HEK293-6E cells yielded about 1 to 2 mg of purifiedprotein per L of day 7 transient transfection CM. FIG. 51 shows thealignment of human and cynomolgus CD163 proteins.

Armed with the knowledge of the HDX-MS results that identify the bindingepitope on CD163 and the significant sequence identity between human andcynomolgus CD163 and that AB101 does not bind cynomolgus CD163, allowedthe identification a key residue in SRCR domain 3 implicated AB101binding. The lysine residue at position 323 in human CD163 is a glutamicresidue in all potential non-human species considered for toxicologystudies (FIG. 51 ). This position is centered within the AB101 bindingepitope of SRCR domain 3 that was defined by HDX-MS. Site-directedmutagenesis of the cynomolgus glutamic acid at position 323 to lysineconfers AB101 binding to cynomolgus CD163 ECD with an EC50 near that ofAB101 binding to human CD163 ECD (FIG. 52 ). This gain in functionresult strongly implicates the lysine at position 323 in AB101 bindingepitope.

Example 25—Binding Affinity of AB101 to Human CD163 by SPR

Surface plasmon resonance (SPR) measurements were used to determineAB101 and anti-CD163 clone GHI/61 binding to human CD163 (AB101 aviditymeasurements) in different running buffer conditions.

SPR analysis of binding of AB101 and anti-CD163 clone GHI/61 to humanCD163 was carried out using a Biacore T200 instrument (GE HealthcareLife Sciences). The purified recombinant CD163 protein was directlyimmobilized on the chip (Series S-type CMS) using an amine coupling kit(GE Healthcare Life Sciences). A pH scouting (10 mM sodium acetate pH5.5/5.0/4.5/4.0) was performed to determine suitable concentration andpH for amine coupling immobilization. The sodium acetate (pH 5.5) waschosen as the best condition for CD163 coupling onto the sensor chipCMS. The amount of CD163 protein coupling onto the CMS chip was 80 RU(0.08 ng/mm²).

The running buffers used for the experiment: Buffer (1) 10 mM Hepes, 150mM NaCl, 3.0 mM EDTA, and 0.05% Tween 20, pH 7.4 or buffer; Buffer (2)10 mM Hepes, 150 mM NaCl, 3.0 mM CaCl₂, 1.0 mM EGTA, and 0.005% Tween20, pH 7.4.

MAbs were dissolved in running buffer. For AB101, sensorgrams weregenerated using flow rate at 30 μl/min, concentrations at6.25/12.5/25/50/100/200 μg/ml, contact time 300 s, and dissociation time600 s. For GHI/61, sensorgrams were generated using flow rate at 30μl/min, concentrations at 3.125/6.25/12.5/25/50/100 μg/ml, contact time300 s, and dissociation time 600 s. The flow cells were regenerated with10 mM Glycine-HCl, pH 3.0 (GE Healthcare Life Sciences). Data analysiswas performed on the Biacore T200 computer and with the Biacore T200evaluation software.

It has been shown that the binding of GHI/61 and other anti-CD163antibodies to human CD163 is dependent on free calcium. GHI/61 hadhigher affinity to CD163 in calcium free buffer than incalcium-containing buffer. To determine if AB101 binding is calciumdependent, we performed SPR measurement of AB101 binding to immobilizedhuman CD163 in 2 mM calcium-containing buffer or calcium free, EDTAbuffer. As shown in Table 11 and FIG. 53 , AB101 had stronger bindingavidity with a K_(D) of 45 nM in calcium-containing buffer as related tothe 2-fold weaker avidity in calcium free EDTA buffer (K_(D)=89 nM). Incontrast, SPR measurements observed K_(D) values of 63 nM and 12 nM forGHI/61 binding to CD163 in calcium-containing and EDTA buffer (FIG. 54and Table 11), respectively.

TABLE 11 SPR Binding Results of AB101 to Immobilized Human CD163 Bindingof Antibodies to Immobilized Human CD163 AB101 GHI/61 K_(D) k_(a) K_(d)K_(D) k_(a) K_(d) Buffer [nM] [M⁻¹s⁻¹] [s⁻¹] [nM] [M⁻¹s⁻¹] [s⁻¹] Calcium45 1.678 × 10⁴ 7.544 × 10⁻⁴ 63 0.732 × 10⁴ 4.668 × 10⁻⁴ EDTA 89 1.280 ×10⁴ 11.39 × 10⁻⁴ 12 1.476 × 10⁴ 1.783 × 10⁻⁴

The SPR results confirmed the previously reported GHI/61 findings andindicated that AB101 may require physiological calcium concentrationsfor optimal binding to human CD163.

Next, we performed SPR measurements of CD163 binding affinity toimmobilized AB101 in calcium-containing buffer and observed a K_(D) of12 nM (FIG. 55 and Table 12). CD163 binding to immobilized AB101 had a3.7-fold higher ka of 6.213×10⁴ (M⁻¹s⁻¹) when compared to thecorresponding Ka of AB101 binding to immobilized CD163 (1.678×10⁴M⁻¹s⁻¹).

TABLE 12 SPR Binding Results of human CD163 to Immobilized AB101 HumanCD163 Binding to AB101 K_(D) k_(a) K_(d) Buffer [nM] [M⁻¹s⁻¹] [s⁻¹]Calcium 12 6.213 × 10⁴ 7.895 × 10⁻⁴

In summary, SPR measurements determined a 12 nM binding affinity and a45 nM avidity of AB101 to CD163 in the presence of 2 mM free calcium.

Example 26—AB101 Binding to Human CD163 Protein in Solution by AlphaLISA

The binding affinity of AB101 to human CD163 in solution was determinedby AlphaLisa assay.

The assay consists of human soluble CD163 with 10-histidine tag (SEQ IDNO: 23) on the C-terminus, biotinylated anti-hIgG1, streptavidinacceptor beads and nickel donor beads.

The assay was performed by making serial dilutions of AB101 and isotypecontrol in 0.5% BSA in 1×PBS without Ca²⁺, Mg²⁺ with a startingtitration concentration of 1500 nM. CD163 was diluted in the same bufferto a concentration of 1500 nM.

Binding of AB101 to human CD163. AB101 or isotype control serialdilutions were added at a volume of 5 μl/well to 5 μl/well human CD163,sealed with aluminum plate sealer and incubated for 1 hour at roomtemperature with gentle shaking.

Detection of AB101: AB101 was detected using 2.5 nM biotinylatedanti-human IgG1 antibody in 1×AlphaLISA Immunoassay assay buffer at avolume of 5 μl/well to the 10 μl binding mix. The plate was sealed andincubated for 1 hour at room temperature with gentle shaking.

Binding of acceptor bead: Following antibody detection step, 5 μl of 100μg/ml streptavidin acceptor beads in 1×AlphaLISA Immunoassay assaybuffer were added to each well, followed by sealing the plate andincubating at room temperature for 1 hour with gentle shaking.

Binding of donor bead: Next, 5 μl of 100 μg/ml nickel donor beads in1×AlphaLISA Immunoassay assay buffer were added per well, followed bysealing the plate and incubating at room temperature for 1 hour withgentle shaking.

The plate was read using an Envision plate reader according tomanufacturer protocol, and data was analyzed using GraphPad Prism 8 tocalculate K_(d).

The AlphaLisa measurement of AB101 binding to CD163 reached saturationat 30 nM of AB101 (FIG. 56 ). The K_(d) of 1.8 nM was determined by a1-site saturated binding model combining 5 independent measurements(Table 13). The 2-site saturated binding model provided a better curvefit for the lower AB101 concentration.

TABLE 13 AlphaLisa Measurements of AB101 Binding to Soluble CD163Protein Binding AB101 to Soluble CD163 1-Site Binding Model IndependentEC₅₀ Assays [nM] Max R² 1 4.1 31000 0.99 2 1.5 37000 0.97 3 1.9 520000.98 4 1.7 48000 0.99 5  0.51 44000 0.98 Mean 1.9 42400 SD 1.3  8444Geometrical Mean 1.6 41692

Example 27—Binding of AB101 to M2c Macrophages

To determine the binding kinetics of AB101 to CD163 expressed on cells,we performed binding studies with immunosuppressive M2c macrophages from4 study subjects.

AB101 binding to M2c macrophages was evaluated from four healthy humansubjects (39-year-old female, 24 year old male, 39 year old male and 54year old male). Monocytes were isolated from LeukoPaks purchased fromBloodWorks.

At Day 7, M2c cells were incubated for 15 minutes at room temperature inMacrophage Detachment Solution DXF and removed from the flask intoX-VIVO-15™ medium. Following centrifugation, the cells were washed oncewith PBS and resuspended in Zombie UV live/dead stain (1:500) at roomtemperature for 20 minutes. Cells were then washed with FACS buffer andresuspended in FACS Block (FACS buffer containing 10% FBS and 0.5 mg/mlhuman IgG1) at room temperature for 20 minutes. Cells in blocking bufferwere transferred to a 384 well plate at 2.5×10⁴ cells/well and titratedantibodies were added directly to each well at 2× final assayconcentration. Cells were incubated with antibodies at room temperaturefor 20 min. Cells were washed three times with FACS buffer, thenresuspended in FACS buffer for acquisition on a Symphony flow cytometer(BD Biosciences).

AB101 binding to M2c macrophages exhibited a bimodal binding curve (FIG.57 ) suggesting that AB101 binding to the Fc receptor may affect bindingto CD163 expressed on M2c cells. The calculated K_(d) values for AB101in 1-site specific saturated binding curves are shown in Table 14. TheK_(d) value for binding of AB101 to CD163 calculated by the 1-site modelwas 7.7 nM with a Bmax of 46103 gMFI (R²=0.91). Two-site curve fitmodelling provided a better curve fit (R²=0.98).

TABLE 14 Equilibrium Binding of AB101 Binding to Human M2c MacrophagesBinding Kinetics of AB101 1-Site Binding Model Human K_(d) Subject IDs[nM] Bmax R² W 7.2 45800 0.93 X 6.6 41700 0.91 Y 8.1 45400 0.91 Z 8.951513 0.93 Mean 7.7 46103 SD 1.0  4051 Geometrical Mean 7.7 45972

Although the present disclosure and its advantages have been describedin detail, it should be understood that various changes, substitutions,and alterations are made herein without departing from the spirit andscope of the disclosure as defined in the appended claims.

What is claimed is:
 1. A pharmaceutical composition, comprising (a) anantibody or an antigen-binding fragment thereof, comprising: (i) a lightchain variable region (VL) comprising, from N-terminus to C-terminus,domains therein having amino acid sequences with 100% identity to thoseof each of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and (ii) aheavy chain variable region (VH) comprising, from N-terminus toC-terminus, domains therein having amino acid sequences with 100%identity to those of each of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:6; and (b) one or more pharmaceutically acceptable excipients.
 2. Thepharmaceutical composition of claim 1, wherein the amino acid sequenceof the VL has at least 85% identity to that of SEQ ID NO: 7 and theamino acid sequence of the VH has at least 85% identity to that of SEQID NO:
 8. 3. The pharmaceutical composition of claim 1, wherein theamino acid sequence of the VL has at least 90% identity to that of SEQID NO: 7 and the amino acid sequence of the VH has at least 90% identityto that of SEQ ID NO:
 8. 4. The pharmaceutical composition of claim 1,wherein the amino acid sequence of the VL has at least 95% identity tothat of SEQ ID NO: 7 and the amino acid sequence of the VH has at least95% identity to that of SEQ ID NO:
 8. 5. The pharmaceutical compositionof claim 1, wherein the amino acid sequence of the VL has at least 99%identity to that of SEQ ID NO: 7 and the amino acid sequence of the VHhas at least 99% identity to that of SEQ ID NO:
 8. 6. The pharmaceuticalcomposition of claim 1, wherein the amino acid sequence of the VL has100% identity to that of SEQ ID NO: 7 and the amino acid sequence of theVH has 100% identity to that of SEQ ID NO:
 8. 7. The pharmaceuticalcomposition of claim 1, wherein the antibody or antigen-binding fragmentthereof further comprises a human heavy chain constant region or a humanlight chain constant region.
 8. The pharmaceutical composition of claim1, wherein the antibody or antigen-binding fragment thereof comprises:a. a light chain having at least 80% identity to the amino acid sequenceof SEQ ID NO: 9; and b. a heavy chain having at least 80% identity tothe amino acid sequence of SEQ ID NO:
 10. 9. The pharmaceuticalcomposition of claim 1, wherein the antibody or antigen-binding fragmentthereof comprises a human variable framework region and a murineconstant region.
 10. The pharmaceutical composition of claim 1, whereinthe antibody or antigen-binding fragment thereof specifically binds toan epitope comprising an amino acid sequence of SEQ ID NO: 18, SEQ IDNO: 19, and/or SEQ ID NO:
 20. 11. The pharmaceutical composition ofclaim 1, wherein the antibody or antigen-binding fragment thereoffurther comprises a constant domain that binds to an Fc receptorexpressed on a macrophage.
 12. The pharmaceutical composition of claim1, wherein the antibody or antigen-binding fragment thereof binds to amacrophage expressing human CD163.
 13. The pharmaceutical composition ofclaim 1, wherein the one or more pharmaceutically acceptable excipientsis selected from the group consisting of: surfactants, excipients thatimprove the stability of the antibody or antibody fragment, water, andcombinations thereof.
 14. The pharmaceutical composition of claim 1,wherein the one or more pharmaceutically acceptable excipients comprisesone or more surfactants.
 15. The pharmaceutical composition of claim 1,wherein the one or more pharmaceutically acceptable excipients comprisesone or more excipients that improve(s) the stability of the antibody orantibody fragment.
 16. A pharmaceutical composition, comprising (a) anantibody or an antigen-binding fragment thereof, comprising: (i) a lightchain variable region (VL) having an amino acid sequence with at least85% identity to that of SEQ ID NO: 7, and comprising, from N-terminus toC-terminus, domains therein having amino acid sequences with 100%identity to those of each of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:3, and (ii) a heavy chain variable region (VH) comprising an amino acidsequence with at least 85% identity to that of SEQ ID NO: 8, andcomprising, from N-terminus to C-terminus, domains therein having aminoacid sequences with 100% identity to those of each of SEQ ID NO: 4, SEQID NO: 5, and SEQ ID NO: 6; and (b) one or more pharmaceuticallyacceptable excipients.
 17. The pharmaceutical composition of claim 16,wherein the amino acid sequence of the VL has at least 90% identity tothat of SEQ ID NO: 7 and the amino acid sequence of the VH has at least90% identity to that of SEQ ID NO:
 8. 18. The pharmaceutical compositionof claim 16, wherein the amino acid sequence of the VL has at least 95%identity to that of SEQ ID NO: 7 and the amino acid sequence of the VHhas at least 95% identity to that of SEQ ID NO:
 8. 19. Thepharmaceutical composition of claim 16, wherein the amino acid sequenceof the VL has at least 99% identity to that of SEQ ID NO: 7 and theamino acid sequence of the VH has at least 99% identity to that of SEQID NO:
 8. 20. The pharmaceutical composition of claim 16, wherein theamino acid sequence of the VL has 100% identity to that of SEQ ID NO: 7and the amino acid sequence of the VH has 100% identity to that of SEQID NO:
 8. 21. The pharmaceutical composition of claim 16, wherein theantibody, recombinant antibody, or antigen-binding fragment thereoffurther comprises a human heavy chain constant region or a human lightchain constant region.
 22. The pharmaceutical composition of claim 16,wherein the antibody or antigen-binding fragment thereof comprises: a. alight chain having at least 80% identity to the amino acid sequence ofSEQ ID NO: 9; and b. a heavy chain having at least 80% identity to theamino acid sequence of SEQ ID NO:
 10. 23. The pharmaceutical compositionof claim 16, wherein the antibody or antigen-binding fragment thereofcomprises a human variable framework region and a murine constantregion.
 24. The pharmaceutical composition of claim 16, wherein theantibody or antigen-binding fragment thereof specifically binds to anepitope comprising an amino acid sequence of SEQ ID NO: 18, SEQ ID NO:19, and/or SEQ ID NO:
 20. 25. The pharmaceutical composition of claim16, wherein the antibody or antigen-binding fragment thereof furthercomprises a constant domain that binds to an Fc receptor expressed on amacrophage.
 26. The pharmaceutical composition of claim 16, wherein theantibody, recombinant antibody, or antigen-binding fragment thereofbinds to a CD163-expressing macrophage.
 27. The pharmaceuticalcomposition of claim 16, wherein the one or more pharmaceuticallyacceptable excipients is selected from the group consisting of:surfactants, excipients that improve the stability of the antibody orantibody fragment, water, and combinations thereof.
 28. Thepharmaceutical composition of claim 16, wherein the one or morepharmaceutically acceptable excipients comprises one or moresurfactants.
 29. The pharmaceutical composition of claim 16, wherein theone or more pharmaceutically acceptable excipients comprises one or moreexcipients that improve(s) the stability of the antibody or antibodyfragment.